Releasable nonvolatile mass-label molecules

ABSTRACT

Releasable tag reagents for use in the detection and analysis of target molecules, particular in mass spectrometric analyses are provided. Also provided are methods of detection that employ releasable tag reagents.

RELATED APPLICATION

[0001] This application is a continuation of U.S. application Ser. No.08/988,024, filed Dec. 10, 1997, to MONFORTE, JOSEPH A., BECKER,CHRISTOPHER H., POLLART, DANIEL J. and SHALER, THOMAS A., entitled“RELEASABLE NONVOLATILE MASS-LABEL MOLECULES.” Benefit of priority under35 U.S.C. §119(e) is also claimed to U.S. provisional application SerialNo. 60/033,037, filed Dec. 10, 1996, and Serial No. 60/046,719, filedMay 16, 1997. The subject matter of each of these applications isincorporated by reference in its entirety.

[0002] The government may own rights in subject matter herein pursuantto Cooperative Agreement No. 70NANB5H1029 from the United StatesDepartment of Commerce, Advanced Technology Program.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to the field of chemicalanalysis. More particularly, it concerns a new class of nonvolatile,releasable tag reagents for use in the detection and analysis of targetmolecules i.e., mass spectrometry.

[0005] 2. Description of Related Art

[0006] Chemical labels, otherwise known as tags or signal groups, arewidely used in chemical analysis. Among the types of molecules used areradioactive atoms, fluorescent reagents, luminescent reagents,metal-containing compounds, electron-absorbing substances and lightabsorbing compounds. Chemical signal groups can be combined withreactivity groups so that they might be covalently attached to thetarget, the substance being detected. In many cases, however, chemicalmoieties present on the target may interfere with the detection of thesignal group or not allow for measurement of the signal group in anoptimal detection environment.

[0007] Indirect detection of the target is oftentimes, therefore,preferred. For example, the signal group may be the product of thedegradation of the target or a derivative of the target (Bueht et. al.,1974; Senft, 1985; U.S. Pat. Nos. 4,650,750; 4,709,016; 4,629,689).Volatile releasable tag compounds that can be analyzed using variousforms of electron-attachment mass spectrometry, often with gaschromatography-mass spectrometry (GC-MS), have been described (Wang etal., 1996; U.S. Pat. Nos. 5,360,819; 5,516,931). Despite the broad rangeof volatile mass labels reported, a transition from liquid to gas phaseis required for analysis which places significant synthetic and sizeparameters on the label. Isotopic mass labels have also been described,such as using tin or sulfur isotopes, with various mass spectrometricsampling approaches (Arlinghaus et al. 1997; U.S. Pat. No. 5,174,962).The isotopic labeling often limits the extent of multiplexing andprovides a more complex analysis requirement.

[0008] Mass spectral analysis of signal groups involves none of theconcerns related to radioactive signal groups, such as their shorthalf-lives and their safety and disposal issues. Another key advantageto detection of signal groups via mass spectrometry is that it allows agreat ability to multiplex, to detect for more than one signal group ina complex mixture, and therefore more than one target at a time. Brummelet al. (1994; 1996) have demonstrated the use of mass spectrometry inthe direct analysis of combinatorial libraries of small peptides.However, use of this technology is limited to analysis of the entirereacting compound by mass spectrometry.

[0009] Detection of multiple fluorescent labels has been used to analyzenucleic acid sequences. Nucleic acid hybridization probes are modifiedto contain fluorescent chromophores that when excited by light emit aunique color spectrum signature. In fluorescence based sequencingsystems, four different chromophores can be multiplexed within a sampleand individually detected with the aid of software deconvolution. Thepractical upper limit for fluorescence multiplexing is likely to bearound 10 different labels due to the broad overlapping spectrumproduced by existing fluorescent chromophores. Clearly the developmentof nonvolatile releasable mass labels, detectable over the usable rangeof a mass spectrometer, would represent a significant advantage bypermitting the multiplexing of tens, hundreds and perhaps even thousandsof different mass labels that can be used to uniquely identify eachdesired target.

[0010] At present, while tools are available through which targetmolecules may be detected, there remains a need for further developmentof these systems in order to analyze a large number of targetssimultaneously. This will allow for the systematic analysis of targetmolecules with predetermined properties and functions.

SUMMARY OF THE INVENTION

[0011] It is, therefore, a goal of the present Invention to providecompositions and methods relating to the use of release tag compoundsfor detection and analysis of target molecules.

[0012] The present invention relates to the use of nonvolatile,releasable tag compounds, containing releasable mass labels, in chemicalanalysis, and to the use of these reagents in conjunction with probeswhich react with or bind noncovalently to a molecule whose presence isto be detected. The releasable tag reagents thus may indirectly detecttarget molecules, including biomolecular targets. The mass label may bereleased from the probe following reaction with or binding of the probeto the target and detected by mass spectrometry. The mass value of thelabel identifies and characterizes the probe and, therefore, the targetmolecule. In the case of a mass-labeled oligonucleotide probe used totarget a polynucleotide, the detection of mass-labels rather than thenucleic acid probes or the nucleic acid targets themselves means thatbiochemical analysis procedures can be greatly simplified. The need forslow, laborious, costly, and/or complex solid-phase and/orsolution-phase cleanup and desalting procedures can be minimized or eveneliminated.

[0013] Therefore, in accordance with the present invention, there isprovided a release tag compound comprising Rx, Re and M, wherein Rx is areactive group, Re is a release group, and M is a mass label detectableby mass spectrometry. As used herein the term “a” encompassesembodiments wherein it refers to a single element as well as embodimentsincluding one or more of such elements. For example, the phrase “areactive group: may refer to a single reactive group, but alsoencompasses embodiments including more than one reactive group.

[0014] Although the mass label may typically be a synthetic polymer or abiopolymer or some combination thereof, in some embodiments, the masslabel may generally be any compound that may be detected by massspectrometry. In particular embodiments, the mass label may be abiopolymer comprising monomer units, wherein each monomer unit isseparately and independently selected from the group consistingessentially of an amino acid, a nucleic acid, and a saccharide withamino acids and nucleic acids being preferred monomer units. Becauseeach monomer unit may be separately and independently selected,biopolymer mass labels may be polynucleic acids, peptides, peptidenucleic acids, oligonucleotides, and so on.

[0015] As defined herein “nucleic acids” refer to standard ornaturally-occurring as well as modified/non-natural nucleic acids, oftenknown as nucleic acid mimics. Thus, the term “nucleotides” refer to bothnaturally-occurring and modified/nonnaturally-occurring nucleotides,including nucleoside tri, di, and monophosphates as well asmonophosphate monomers present within polynucleic acid oroligonucleotide. A nucleotide may also be a ribo; 2′-deoxy; 2′, 3′-deoxyas well as a vast array of other nucleotide mimics that are well-knownin the art. Mimics include chain-terminating nucleotides, such as3′-O-methyl, halogenated base or sugar substitutions; alternative sugarstructures including nonsugar, alkyl ring structures; alternative basesincluding inosine; deaza-modified; chi, and psi, linker-modified; masslabel-modified; phosphodiester modifications or replacements includingphosphorothioate, methylphosphonate, boranophosphate, amide, ester,ether; and a basic or complete internucleotide replacements, includingcleavage linkages such as photocleavable nitrophenyl moieties. Thesemodifications are well known by those of skill in the art and based onfundamental principles as described Saenger (1983), incorporated hereinby reference.

[0016] Similarly, the term “amino acid” refers to naturally-occurringamino acid as well as any modified amino acid that may be synthesized orobtained by methods that are well known in the art.

[0017] In another embodiment, the mass label may be a synthetic polymer,such as polyethylene glycol, polyvinyl phenol, polyproplene glycol,polymethyl methacrylate, and derivatives thereof. Synthetic polymers maytypically contain monomer units selected from the group consistingessentially of ethylene glycol, vinyl phenol, propylene glycol, methylmethacrylate, and derivatives thereof. More typically the mass label maybe a polymer containing polyethylene glycol units.

[0018] The mass label is typically detectable by a method of massspectrometry. While it is envisioned that any known mass spectrometrymethod may be used to detect the mass labels of the present invention,methods such as matrix-assisted laser-desorption ionization massspectrometry, direct laser-desorption ionization mass spectrometry (withno matrix), electrospray ionization mass spectrometry, secondary neutralmass spectrometry, and secondary ion mass spectrometry are preferred.

[0019] In certain embodiments the mass label has a molecular weightgreater than about 500 Daltons. For some embodiments, it may bepreferred to have nonvolatile (including involatile) mass labels,however, for other embodiments volatile mass labels are alsocontemplated.

[0020] As defined herein, the term “reactive group” refers to a groupcapable of reacting with the molecule whose presence is to be detected.For example, the reactive group may be a biomolecule capable of specificmolecular recognition. Biomolecules capable of specific molecularrecognition may typically be any molecule capable of specific bindinginteractions with unique molecules or classes of molecules, such aspeptides, proteins, polynucleic acids, etc.

[0021] Thus, reactive groups disclosed herein for use with the disclosedmethods encompass polypeptides and polynucleic acids. As used herein,polypeptides refer to molecules containing more than one amino acid(which include native and non-native amino acid monomers. Thus,polypeptides includes peptides comprising 2 or more amino acids; nativeproteins; enzymes; gene products; antibodies; protein conjugates; mutantor polymorphic polypeptides; post-translationally modified proteins;genetically engineered gene products including products of chemicalsynthesis, in vitro translation, cell-based expression systems,including fast evolution systems involving vector shuffling, random ordirected mutagenesis, and peptide sequence randomization. In preferredembodiments polypeptides may be oligopeptides, antibodies, enzymes,receptors, regulatory proteins, nucleic acid-binding proteins, hormones,or protein product of a display method, such as a phage display methodor a bacterial display method. More preferred polypeptide reactivegroups are antibodies and enzymes. As used herein, the phrase “productof a display method” refers to any polypeptide resulting from theperformance of a display method which are well known in the art. It iscontemplated that any display method known in the art may be used toproduce the polypeptides for use in conjunction with the presentinvention.

[0022] Similarly, “polynucleic acids” refer to molecules containing morethan one nucleic acid. Polynucleic acids include lengths of 2 or morenucleotide monomers and encompass nucleic acids, oligonucleotides,oligos, polynucleotides, DNA, genomic DNA, mitochondrial DNA (mtDNA),copy DNA (cDNA), bacterial DNA, viral DNA, viral RNA, RNA, message RNA(mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), catalytic RNA,clones, plasmids, M13, P1, cosmid, bacteria artificial chromosome (BAC),yeast artificial chromosome (YAC), amplified nucleic acid, amplicon, PCRproduct and other types of amplified nucleic acid. In preferredembodiments, the polynucleic acid may be an oligonucleotide.

[0023] In still further embodiments, Rx is an oligonucleotide having oneor more nucleotides or oligonucleotide is added after hybridization ofRx to a complementary nucleic acid sequence. The term complementarygenerally refers to the formation of sufficient hydrogen bonding betweentwo nucleic acids to stabilize a double-stranded nucleotide sequenceformed by hybridization of the two nucleic acids.

[0024] Typically, nucleotides may be added by a polymerase whileoligonucleotides may be added by a ligase. However, it is alsocontemplated that other methods of adding nucleotides andoligonucleotides known by those of skill in the art may also beemployed. In further embodiments, it is provided that the nucleotideadded after hybridization may have a chain terminating modification, forexample, the added nucleotide may be a chain terminating dideoxynucleotide.

[0025] Embodiments are also provided wherein the added nucleotide oroligonucleotide further comprise a functional group capable of beingimmobilized on a solid support, for example, a biotin or digoxigenin.Generally, this functional group or binding group or moiety is capableof attaching or binding the tag compound to the solid support. Thisbinding moiety may be attached to the added nucleotide oroligonucleotide directly through an intervening linking group or byspecific hybridization to an intermediary oligonucleotide which isitself bound to a solid support. Binding moieties include functionalgroups for covalent bonding to a solid support, ligands that attach tothe solid support via a high-affinity, noncovalent interaction (such asbiotin with streptavidin), a series of bases complementary to anintermediary oligonucleotide which is itself attached to the solidsupport, as well as other means that are well-known to those of skill inthe art, such as those described in PCT WO 96/37630, incorporated hereinby reference.

[0026] In other embodiments, the reactive group may contain a nucleaseblocking moiety. These moieties serve to block the digestion of theoligonucleotide by the nuclease, such as an exonuclease. Typicalnuclease blocking moieties thus include phosphorathioate,alkylsilyldiester, boranophosphate, methylphosphonate, and peptidenucleic acid.

[0027] The mass label is linked, or attached, to the reactive group viaa releasable attachment. Thus, typically the mass label is released fromall or a part of the reactive group prior to mass spectral analysis ascontemplated by the various methods described herein. This releasableattachment typically occurs through the use of a release group which maybe the linkage between the mass label and the reactive group or whichmay comprise a portion or all of the reactive group or which may becontained within the reactive group.

[0028] The release group may be any labile group providing for such areleasable attachment. The release group may thus be a chemicallycleavable linkage or labile chemical linkage. Such linkages maytypically be cleaved by methods that are well known to those of skill inthe art, such as by acid, base, oxidation, reduction, heat, light, ormetal ion catalyzed, displacement or elimination chemistry. In aparticular embodiment, the chemistry cleavable linkage comprises amodified base, a modified sugar, a disulfide bond, a chemicallycleavable group incorporated into the phosphate backbone, or achemically cleavable linker. Some examples of these linkages aredescribed in PCT WO 96/37630, incorporated herein by reference. As usedherein, “chemically cleavable linkers” are moieties cleavable by, forexample, acid, base, oxidation, reduction, heat, light, metal ioncatalyzed, displacement or elimination chemistry.

[0029] Chemically cleavable groups that may be incorporated into thephosphate backbone are well known to those of skill in the art and mayinclude dialkoxysilane, 3′-(S)-phosphorothioate,5′-(S)-phosphorothioate, 3′-(N)-phosphoroamidate, or5′-(N)-phosphoroamidate. In further embodiments the chemically cleavablelinkage may be a modified sugar, such as ribose. Alternatively, thelinkage may be a disulfide bond.

[0030] In still yet another embodiment, Re is contained within Rx. Inthis case, the release of Re may be activated by a selective event. Inparticular embodiments, the selective release is mediated by an enzymesuch as an exonuclease specific for double-stranded or single-strandedDNA. When it is said that Re is contained within Rx, it will generallybe understood that the reactive group contains within its structure theparticular release group which will cause the mass label to disconnectfrom the tag compound in that particular embodiment.

[0031] Thus, release groups encompassed by the invention also includegroups or linkages cleavable by an enzyme. Enzymatically-cleavablerelease groups include phosphodiester or amide linkages as well asrestriction endonuclease recognition sites.

[0032] Preferred embodiments encompass release groups cleavable bynucleases. These nucleases may typically be an exonuclease or arestriction endonuclease. Typical exonucleases include exonucleasesspecific for both double-stranded and single-stranded polynucleic acids.Additionally, restriction endonucleases encompassed by certainembodiments include Type IIS and Type II restriction endonucleases.

[0033] In other embodiments the release group may be cleavable by aprotease. Typical proteases include endoproteinases.

[0034] Also provided are embodiments wherein Rx comprises a nucleosidetriphosphate or is synthesized using mass-labeled nucleosidetriphosphates. In another embodiment, Rx comprises a nucleosidephosphoramidite or is synthesized using mass-labeled nucleosidephosphoramidites.

[0035] In still further embodiments, mass-labeled probes are providedwherein at least one component is a nucleoside triphosphate. It isfurther contemplated that the labeled probes of the invention mayinclude at least two unique mass-labels are incorporated.

[0036] Also provided are release tag compounds comprising Rx, Re and M,wherein Rx is a double-stranded oligonucleotide comprising a restrictionendonuclease recognition site; Re is a release group comprising aphosphodiester linkage capable of being cleaved by a restrictionendonuclease; and M is a mass label detectable by mass spectrometry. Rxmay further include a modified nucleotide and the mass label may includea portion of Rx.

[0037] Double-stranded oligonucleotides as provided herein include notonly two complementary strands hybridized to each other via hydrogenbonding interactions, but also include single strands of nucleotideswherein portions of the strand are single-stranded and portions aredouble-stranded. For example, portions or all of Rx may include aself-complementary oligonucleotide hairpin where part of Rx iscomplementary to another part of Rx. In this case, certain conditionsallow the formation of a double-stranded duplex between these twoportions of Rx. For purposes of certain embodiments of the presentinvention, it is not necessary that all of Rx need be double-stranded,release tag compounds containing single-stranded regions are alsocontemplated as being within this embodiment.

[0038] Release tag compound are also contemplated having Rx, Re and M,wherein: Rx is a double-stranded oligonucleotide; Re is a chemicallycleavable release group and M is a mass label detectable by massspectrometry. In this embodiment, Re is typically located within Rx.Cleavage at the chemically cleavable release group is generallyinhibited in this aspect by the presence of a double-strandedoligonucleotide at the release group. Previously discussed chemicallycleavable release groups, such as 3′-(S)-phosphorothioate,5′(S)-phosphorothioate, 3′-(N)-phosphoroamidate,5′-(N)-phosphoroamidate, or ribose, may be employed with theseembodiments. In these embodiments, a portion of Rx may be renderedsingle-stranded at Re by hybridization of a portion of Rx to a targetnucleic acid.

[0039] Also provides is a set of release tag compounds for detecting aparticular target nucleic acid. In this aspect, the target nucleic acidtypically contains more than one release tag compound. Each release tagcompound includes the elements Rx, Re and M, where Rx is anoligonucleotide including a variable region and an invariant region; Reis a release group; and M is a mass label detectable by massspectrometry. The invariant and variable regions react with the targetnucleic acid. It will generally be understood by those of skill in theart that the term “set” refers to a group of two or more release tagcompounds. Generally each member, i.e., each release tag compound of thegroup will be different from all other members of the group. That is,each member will include a different combination of reactive grouprelease group and mass label.

[0040] Typically, the mass label of at least one member of the set mayidentify a specific sequence within the variable region. In someembodiments, the mass label for each member of the set may uniquelyidentify each different sequence within the variable region. In otherembodiments, a combination of the mass labels of two or more release tagcompounds may identify each different sequence within the variableregion.

[0041] As previously discussed, Rx may further comprise a nucleotide oroligonucleotide added after hybridization to the target nucleic acid. Inthis aspect, the added nucleotide or oligonucleotide may furthercomprise Re′ and M′, where Re′ is a release group; and M′ is a masslabel detectable by mass spectrometry. The added nucleotide oroligonucleotide may also contain a chain terminating moiety or afunctional group capable of being immobilized on a solid support, suchas biotin or digoxigenin.

[0042] Methods of producing a mass-labeled probe are provided,comprising combining nucleoside or amino acid monomers with at least onemass-labeled monomer under conditions to allow for polymerization.

[0043] Further embodiments are provided wherein the polymerization ismediated by an enzyme. Still further embodiments are provided whereinthe polymerization is mediated by chemical synthesis. The preferredsynthetic methods to prepare the compound of the present invention areessentially those for standard peptide and DNA synthesis.

[0044] For particular embodiments, synthesis in the solid phase ispreferred to allow for a wide variety of compounds to be produced usingcombinatorial methods.

[0045] Additional embodiments are provided for a method of producing amass-labeled probe, comprising the steps of (a) combining nucleosidemonomers with at least one activated nucleoside monomer under conditionsto allow for polymerization; and (b) adding a releasable, nonvolatilemass unit to said activated nucleoside monomer.

[0046] The present invention also provides embodiments which provide amethod for detecting a target molecule. Generally, the method includesobtaining a plurality of probes, each probe including a reactive group,a release group and a mass label, as described. It is preferred thateach probe within the plurality contains a unique mass-label. By “uniquemass label” it is meant that each probe within the plurality will have adifferent mass label from all other probes in the plurality. A pluralitywill generally be understood to include two or more probes. Next, thetarget molecule is contacted with the plurality of probes underconditions suitable to allow for the formation of probe: target moleculecomplexes. The mass-label is released from the probe and the mass of themass-label is determined. Typically, the mass is indicative of aspecific target molecule. In this way, the target molecule can beidentified according to the unique combination of mass-labels.

[0047] In another aspect, the invention provides a method for detectinga target molecule where the target molecule is amplified to produce anamplified target molecule. The amplified target molecule is thenhybridized with a probe such as those described hereinabove to produceprobe: amplified target molecule complexes. The mass label on the probeamplified target molecule complexes are then released and the mass ofthe mass label determined by mass spectrometry.

[0048] The target nucleic acid may be amplified by any method known byone of skill in the art, for example, polymerase chain reaction (“PCR”),with PCR being a preferred amplification method. The amplification mayinclude a functional group capable of being immobilized on a solidsupport, such as biotin or digoxigenin. This functional group may beattached to an oligonucleotide primer incorporated into the amplifiedmolecule during the amplification step or it may be attached to anucleotide incorporated into the amplified target molecule during theamplification step.

[0049] Methods are also provided wherein the amplified target moleculeis immobilized onto a solid support and any probe not part of aprobe:amplified target molecule complex is removed by washing. It willbe understood by those of skill in the art that the nature of therecognition of the target molecule by the reactive group will depend onthe identity of the target molecule and the reactive group. For purposesof exemplification and not limitation, this recognition may encompassthe formation of a double-stranded duplex by hybridization where thereactive group and target molecule are oligonucleotides. The mass labelmay be released enzymatically or chemically.

[0050] It is contemplated that useful enzymes for this embodiment willinclude nucleases, such as Type II and IIS restriction endonuclease andexonucleases. The envisioned exonucleases may be specific fordouble-stranded DNA, such as exonuclease III, T4 endonuclease VII,lambda exonuclease, and DNA polymerase. For these embodiments therelease of the mass label may be triggered by the hybridization of theprobe to the amplification product. In that embodiment the probe wouldbe single-stranded and capable of hybridizing to the target whosepresence was to be detected. The exonuclease may also be specific forsingle-stranded DNA.

[0051] chemically cleavable linkages may comprise a modified base, amodified sugar, a disulfide bond, a chemically cleavable groupincorporated into the phosphate backbone, or a chemically cleavablelinker and are typically cleaved by acid, base, oxidation, reduction,heat, light, or metal ion catalyzed, displacement or eliminationchemistry.

[0052] Embodiments are provided wherein the reactive group furthercomprises a nucleotide or oligonucleotide added after hybridization tothe amplification product, amplified target molecule or amplifiednucleic acid molecule. These added nucleotides or oligonucleotides mayoptionally include a functional group capable of being immobilized on asolid support.

[0053] For embodiments employing immobilization onto a solid support,one will typically immobilize the reactive group onto the solid supportafter addition of the nucleotide or oligonucleotide then any probeshaving unbound reactive groups are removed prior to releasing the masslabel of any probe belonging to a probe:amplified target moleculecomplex or probe:target molecule complex.

[0054] In these embodiments, the reactive and release groups may be thesame or the release group may be contained within the reactive group.The probe may also comprise at least two unique mass labels.

[0055] Multiplexing methods are also provided wherein the targetmolecule is contacted with a plurality of probes. In these instances,each reactive group of the probe may be associated with a unique masslabel or it may be associated with a unique set of mass labels. Thus, atarget molecule may be detected by the mass spectral detection of aparticular mass label or a particular set of mass labels. Where a set ofmass labels is employed, the set of mass labels may be attached to thesame probe. Alternatively, each member of the set may be attached to adifferent probe.

[0056] Also provided are methods for detecting mismatches wherein theamplified nucleic acid product comprises a double-stranded moleculecontaining a mismatch, and an exonuclease-blocking functionality at the3′ ends of the strands. Typically, this method may further comprisecleavage of at least one strand of the double-stranded molecule at thesite of the mismatch; and selective releasing of the mass label.Selective releasing of the mass label may typically be accomplished bydigestion of the cleaved strand by a 3′ to 5′ exonuclease, such asexonuclease III.

[0057] As used herein, the term “selective releasing” comprises to thereleasing of a mass label from a probe which belongs to a probe:targetmolecule complex without releasing a mass label from a probe notbelonging to such a complex without having to physically partition thetwo types of probes. However, some embodiments may include bothselective releasing and physical partitioning. The describedimmobilization and washing techniques exemplify a method of physicalpartitioning.

[0058] The mismatch may be cleaved by an enzyme, such as mutHLS, T4endonuclease VII, mutY DNA glycosylase, thymine mismatch DNAglycosylase, or endonuclease V. The mismatch may also be cleaved by achemical, such as OsO₄, HONH₂, or KMnO₄.

[0059] The invention further provides a method for detecting a targetmolecule including the steps of: (a) obtaining a probe including areactive group, a release group and a nonvolatile mass label; (b)contacting a target molecule with the probe to produce probe:targetmolecule complexes; (c) the selectively releasing the mass label fromthe probe:target molecule complexes to produce released mass labels; and(d) determining the mass of the released mass labels by massspectrometry.

[0060] Typically, similar chemical and enzymatic release methods may beemployed with these embodiments. Selective release of the mass label mayalso be accomplished by employing cleavage means that are inhibited bythe presence of a double-stranded oligonucleotide at the said releasegroup. As used in this context, “at said release group” means that basepairing is maintained on both sides of the release group by at least onenucleotide.

[0061] In this embodiment, contacting the probe with the target moleculetypically results in the release group being present in asingle-stranded region because one strand of the probe interacts withthe target molecule, for example, by hybridizing to it.

[0062] Another aspect of the invention encompasses a method formultiplexing the detection of a target molecule including: (a) obtaininga plurality of probes, each probe including a reactive group, a releasegroup and a mass label; (b) contacting the target molecule with theplurality of probes to produce probe:target molecule complexes; (c)releasing the mass label from any probe belonging to probe:targetmolecule complexes to produce released mass labels; and (d) determiningthe mass of any released mass label by mass spectrometry. In thisaspect, each reactive group recognizing a specific target molecule isassociated with a unique set of mass labels. It may often be preferredthat a plurality of target molecules with the plurality of probes.

[0063] The members of the set of mass labels may be attached to the sameprobe or to different probes. Additionally, the same mass label may be amember of sets identifying more than one reactive group. Thus, in thisembodiment the set of mass labels, and not the individual mass label, isunique to a particular reactive group. In this embodiment, probes havinga reactive group that identifies a particular target may vary in releasegroup and mass label as well as in other respects.

[0064] Immobilization and washing techniques may be employed with thisembodiment and it may be preferred in some embodiments to immobilize aplurality of target molecules onto the solid support at spaced locationsand to then contact them with the mass-labeled probes. Typical targetmolecules include a polynucleotide, an antigen, a ligand, a polypeptide,a carbohydrate, and a lipid.

[0065] In further embodiments it may be preferred to employ sets of masslabels wherein a mass label member of the set represents a particularmoiety or functionality or subset of the target molecule. For example,mass label A could correspond to a reactive group composed of A′X₂. . .X_(N) functionalities where A can be anywhere in the reactive group andonly represents A′ and may or may not be structurally related to A′ inany way. Thus, detecting mass label results in the detection of a targetmolecule that recognizes A′, but does not necessarily identify anythingelse about that structure or composition of the target molecule.

[0066] Thus, methods are provided wherein the unique set of mass labelscomprises a mass label that indicates the presence of a specifiedcomponent within the reactive group. Further embodiments also includemethods wherein the mass label indicates the presence of the specifiedcomponent at a specified location within the reactive group. A reactivegroup comprising n specified components may be associated with a uniqueset of mass labels having n members where n may typically be from 1 to1000. Generally, mass labels are individually attached to the reactivegroup and are identified intact.

[0067] A reactive group comprising n specified components may also beassociated with a unique set of mass labels having y members wherein nis less than y!/[x!(y−x)!]; and wherein x comprises the number of masslabels per reactive group.

[0068] In some embodiments a plurality of probes may each comprise aknown reactive group having a known set of mass labels and the pluralityof probes may be prepared by combinatorial synthesis. The plurality oftarget molecules may also comprise a known chemical structure.

[0069] Also provided is a method of monitoring gene expression including(a) obtaining a plurality of probes, each including a reactive group, arelease group and a mass label; (b) contacting a plurality of targetnucleic acids with the plurality of proves to produce probe:targetnucleic acid complexes; (c) selectively releasing the mass label fromany probe belonging to a probe:target nucleic acid complexes to producereleased mass labels; and (d) determining the mass of any released masslabel by mass spectrometry.

[0070] Typically, the target nucleic acids may have sequencesrepresentative of the genes being expressed in a particular cell cultureand are present in concentrations related to their mRNA abundancelevels. The target nucleic acids may typically comprise mRNA orfirst-strand cDNA as well as amplified nucleic acid products.

[0071] Such amplified nucleic acid products may be produced using PCR,rtPCR, LCR, Qbeta Replicase, SDA, CPR, TAS, NASBA, or multiple rounds ofRNA transcription of some combination thereof. Amplification may be usedto selectively amplify a subset of the mBNA pool increasing detectionsignal for these gene products and reducing background from geneproducts outside of the amplified subset.

[0072] Another embodiment encompasses a method of monitoring geneexpression including amplifying a subset of an MRNA pool to produce aplurality of amplified nucleic acid products; contacting a plurality ofamplified nucleic acid products with a plurality of probes, each probeincluding a reactive group, a release group and a mass label to produceprobe:amplified nucleic acid product complexes selectively releasing themass label from any probe belonging to a probe:amplified nucleic acidproduce complexes to produce released mass labels determining the massof any released mass label by mass spectrometry.

[0073] For this embodiment, one more probes or amplified nucleic acidproducts may be capable of being immobilized onto a solid support.

[0074] Another aspect of the invention is a method for detecting atarget molecule, including contacting a target molecule with a probeincluding a reactive group, a release group and a nonvolatile mass labelto produce probe:target molecule complexes; releasing the mass labelfrom any probe belonging to a complex to produce released mass labels;selectively desorbing the released mass label from the mass spectralmatrix such that the probes not belonging to probe:target moleculecomplexes do not desorb; and determining the mass of the released masslabel by mass spectrometry.

[0075] For these embodiments, the mass label should desorb moreefficiently from the mass spectral matrix than the probe or themass-labeled probe. Preferred mass spectral matrices include2,5-dihydroxybenzoic acid, sinapinic acid, oralpha-cyano-4-hydroxycinammic acid.

[0076] A method for detecting a target molecule is also provided. Thismethod includes amplifying one or more target nucleic acids to produceamplified nucleic acid products; incorporating one or more moleculesincluding a reactive group, a release group and a nonvolatile mass labelinto the amplified nucleic acid product during the amplificationprocess; selectively releasing the mass labels incorporated into theamplified nucleic products to produce released mass labels; anddetermining the mass of the released mass labels by mass spectrometry.

[0077] Incorporated molecules may be oligonucleotide primers andnucleoside triphosphates and the amplified nucleic acid products areproduced using PCR, rtPCR, LCR, Qbeta Replicase, SDA, CPR, TAS, NASBA,or multiple rounds of RNA transcription or some combination thereof. Oneor more second molecules, each including a functional group capable ofbeing immobilized on a solid support, may also be incorporated into theamplified nucleic acid products. The functional group may also be usedto bind the amplified nucleic acid products to a solid support, andseparate incorporated mass labeled molecules from unincorporated masslabeled molecules. It may also be preferable to separate the amplifiednucleic acid products from the unincorporated mass labeled molecules,for example, by binding the amplified nucleic acid products to a solidsupport or by hybridizing the amplified nucleic acid products to apolynucleotide bound to solid support. In the latter case, the boundpolynucleotide may be an oligonucleotide, a polyribonucleotide, aplasmid, an M13, a cosmid, a P1 clone, a BAC or a YAC. A plurality ofthese polynucleotides may also be immobilized onto the solid support atspaced locations.

[0078] Also provided is a method for detecting the presence of a targetnucleic acid molecule, said method comprising: obtaining a probecomprising a reactive group, a release group and a mass label;contacting the probe to a target nucleic acid molecule to produceprobe:nucleic acid molecule complexes; mass modifying the probe:nucleicacid molecule complexes by attaching a nucleotide or oligonucleotide tothe probe to produce mass modified mass labels; releasing the massmodified mass labels; and determining the mass of the mass-modified masslabels by mass spectrometry.

[0079] Another embodiment encompasses a method for detecting specificbiomolecules in an enzyme-linked affinity assay comprising: obtaining asubstrate; contacting a target molecule with an affinity ligand-enzymeconjugate to produce an affinity ligand-enzyme conjugate:target moleculecomplex with the substrate to produce a mass modified product; anddetermining the mass of the mass modified product by mass spectrometry.

[0080] As used herein, “affinity ligands” are groups, molecules, ormoieties having an affinity for, or reacting with a particular targetmolecule, similar to the reactive groups employed with the mass labelprobes disclosed above. The affinity ligand may be a biomolecule capableof specific molecular recognition, such as a polypeptide or polynucleicacid. Preferred polypeptides include antibodies, enzymes, receptors,regulatory proteins, nucleic acid-binding proteins, hormones, andprotein products of a display method, such as products of a phagedisplay method or a bacterial display method.

[0081] The enzymes conjugated to these affinity ligands may be anyenzyme that catalyze the conversion of the substrate to a product havinga different mass, such as restriction endonucleases and proteases. Thus,the mass of the substrate has been modified in the production of theproduct by the enzyme. Affinity ligand-enzyme conjugates are moleculeswhere the affinity ligand and enzyme have been attached by the formationof covalent or noncovalent interactions, including hydrogen bonds.

[0082] In some embodiments it may be preferable to employ a plurality ofrestriction endonucleases. In these cases, the various endonucleases maybe conjugated to the affinity ligand to form several affinityligand-enzyme conjugates which are then contacted with the targetmolecule. Similarly, it may be preferable to employ a plurality ofaffinity ligand-enzyme conjugates having different affinity ligands,enzymes, or both.

[0083] The substrate may be any molecule whose conversion to amass-modified product is accomplished by the enzyme employed such as apolypeptide. For embodiments employing restriction endonucleases, it maytherefore comprise a restriction site.

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0085]FIG. 1A and FIG. 1B show generalized examples of two mass-labeledbuilding blocks for the preparation of mass-labeled polynucleotides, amass-labeled nucleoside triphosphate (FIG. 1A) and a mass-labelednucleoside phosphoramidite (FIG. 1B). In these FIGS., B refers to abase, R to an optional releasing linkage, and M to a mass label. Masslabels may also be added after polynucleotide synthesis via linkerreagents.

[0086]FIG. 2A and FIG. 2B show examples of a mass-labeled probe wherethe releasable group is contained within the reactive group and thereleased mass-label includes one or more monomers of the reactive group.

[0087] Shown in FIG. 2A is the use of the probe as an oligonucleotideprimer that can be extended (Step A) by polymerase using nucleosidetriphosphates, including deoxy and dideoxyribonucleotide or combinationsthereof, or by ligase using oligonucleotides. Ligase may be used toattach oligonucleotides to the 5′ as well as the 3′ end. Nucleotides andoligonucleotides added as well as nucleotide monomers within the probemay optionally consist of modified nucleotides or non-natural, mimicnucleotides. Also shown is the optional use of a solid-phase bindinggroup such as biotin (labeled B) that can be used to capture theextended mass-labeled primer prior to release of the mass-label product(Step B). Following release the mass-labeled product is analyzed by massspectrometry (Step C). The non-reactive group component of the masslabel is indicated by Mx, where the x signifies that this component mayhave a single molecular mass or it may represent a combination of 2 ormore molecules of defined mass. The Mx component may be optionallycontained fully within the reactive group and may be comprised ofnucleotides or non-natural, mimic nucleotides. Determining the mass ofthe mass-label product provides the means for identifying the nucleotidecomposition and sequence of bases immediately adjacent to the probe.

[0088]FIG. 2B illustrates the specific case where the mass-labeled probefunctions as a primer to detect a single nucleotide polymorphism. InStep A, following hybridization to a template nucleic acid, a polymeraseis used to add a single nucleotide chain terminator or mass-modifiedversion thereof, selecting from the four possible bases. Following probeextension, the mass-labeled product is released (Step B) and analyzed bymass spectrometry (Step C). As in FIG. 2A, the probe optionallycomprises a solid-phase binding group that may be used to bind and washthe probe prior to the releasing step. In this example a T chainterminator is added increasing the mass of the mass-label product by 298Da, indicating the presence of an A within the template at the targetedposition.

[0089]FIG. 2C illustrates a different embodiment for the use of amass-labeled probe in the determination of single nucleotidepolymorphisms. A mass-labeled probe is hybridized to a template and isextended by polymerase which incorporates a single chain-terminatingnucleotide (Step A). The chain terminating nucleotide is modified tocontain a solid-phase binding group such as biotin (labeled B) that isused to capture the extended mass-labeled primer prior to release of themass-label product (Step D). In this particular illustration the probeis being used to identify whether or not an A nucleotide is present inthe position adjacent to where the probe hybridizes. While the reactionmay include all four chain terminating nucleotides, only the T chainterminator is modified to carry a solid-phase binding group. Thereforeonly if T incorporates, and A is present in the template, will themass-labeled probe be modified and captured to the solid phase (Step B).Use of a Washington step (Step C) prior to release (Step D) will removeany probes that have not incorporated T, removing their mass labels fromthe system. Only probes that were bound to the solid phase (Step B) willbe detected in the mass spectrometer (Step E). The mass label isindicated by Mx, where the x signifies that this component may have asingle molecular mass or it may represent a combination of 2 or moremolecules of defined mass. A multiplex of many different probes ispossible. The release group, Re, may be placed in the linker connectingthe mass label to the probe, or at any position within the backbone ofthe probe. This methodology may be extended to cases where a combinationof nucleotides and chain-terminating nucleotides are used, as well asoligonucleotides, where particular components are selected to contain asolid-phase binding group.

[0090]FIG. 3A and FIG. 3B illustrate a generalized scheme to produce amixture of nucleic acid probes each with a unique single or combinationof mass labels (FIG. 3A) and, in particular, a generalized scheme toincorporate mass-labeled nucleotides or oligonucleotides into apolynucleotide sequence using DNA polymerase (Step A) or ligase (Step B(FIG. 3B).

[0091]FIG. 3A illustrates a nucleic acid probe containing an invariantregion and a variable region. The invariant region, which is optional,carries the same or near the same sequence for all probes within afamily. The variable region contains all possible sequences or somesubset thereof. As an example, if the variable region is 4 nucleotidesin length 256 different probes can be made, if the variable region is 6nucleotides in length 4096 different probe can be made. Associated witheach probe sequence is a single or combination of mass labels. In eithercase, the mass labels chosen are unique to each sequence. In cases wherecombinations are used the mass labels (labeled M) may be single labelsattached to different probes carrying the same sequence or multiplelabels attached to a single probe, or some combination thereof.

[0092]FIG. 3B illustrates two embodiments where the mass-labeled familyof probes may be used to screen a nucleic acid template. In addition tosimple hybridization of the probe to template, the probes may beextended using either polymerase (Step A) or ligase (Step B). In eithercase nucleotides or oligonucleotides may be used that carry additionalmass labels (labeled M*) identifying the sequence of the nucleic acidproduct being added, therefore enlarging the total template sequencedetermined per probe hybridization event. In a preferred embodiment thetemplate is bound to the solid phase. Alternatively, the nucleotides oroligonucleotides added to the probe may contain a solid-phase bindinggroup, enabling the isolation of the probe and attachment viasolid-phase capture. As illustrated, X-Y represents Watson-Crick basepairing in the variable region of the probe, and N-M representsWatson-Crick base pairing in the added nucleotide or oligonucleotidesequence.

[0093]FIG. 4A, FIG. 4B, and FIG. 4C illustrate different combinatorialapproaches to preparing mass labeled probes (FIG. 4A), usingmass-labeled probes to screen a vector insert (FIG. 4B), and enzymaticmethods, including transcription and PCR for the preparation of largemass-labeled polynucleotide probes (FIG. 4C).

[0094]FIG. 4A describes an example of how combinatorial labels may beused to label a complex set of oligonucleotides. The example describes aset of probes that have a variable region 4 nucleotides long comprising256 possible sequence combinations. Variable regions shorter or longerare also possible. In the table and example list (C), it is shown how aset of 16 different mass labels may be used to create a mass labelsignature that is unique for all 256 combinations. Two differentapproaches may be used to creating the labeled probes, the first (A)being the use of 16 different phosphoramidites each containing adifferent mass label that are used according to the base and position ofsynthesis. This approach leads to a set of molecules each with 4 labelson them and is performed as a single reaction. Variants are possiblewhere the synthesis is split into multiple pots and standardphosphoramidite are used in some positions to reduce the number oflabels per molecule. The second combinatorial approach (B) is topresynthesize the 256 combinations in 16 different reactions prior toadding the mass labels, each of which is used to define one of the 4bases in one of the 4 positions. Following oligonucleotide synthesis,each of the 16 different reactions is coupled to one of 16 differentmass labels. The end product is that each probe in the pool containsonly one specific mass label. The second approach offers greaterflexibility for the placement and type of the mass label since it is notcoupled directly to the oligonucleotide synthesis. Other labelingschemes can be envisioned when using the post oligonucleotide synthesismethod especially when the oligonucleotide set is synthesized in alarger number of reactions, with ultimate flexibility if the 256combinations are all synthesized separately. With either approach thesynthesis may optionally include an invariant synthetic region as shownin FIG. 4A. The variable region may also include one or morediscontinuous bases within the invariant region. These probes may beapplied to screening for polymorphisms in diagnostic and genomicapplications including single nucleotide polymorphisms where thevariable region is only one nucleotide long.

[0095]FIG. 4B describes how the combinatorially labeled probes may beused to screen polymorphic sequences that are adjacent to the insertsequences within cloning vectors (A), including cDNA and genomic clones.The use of an invariant sequence within the probes allows the probes tobe anchored at the junction between the known vector sequence and theunknown insert sequence with the invariant region of the probehybridizing to the known sequence and the variable region selecting itscomplement in the unknown region (B). Methods utilizing these probesinclude simple hybridization to one or both of the clone insert ends,nucleotide or oligonucleotide extensions as described in FIG. 3B, anduse of the probes for primer extension to make a single copy of theinsert or for purposes of amplification. For a given insert sequence,use of forward and reverse probes in a PCR amplification would result inthe selection of only one forward and one reverse probe out of the setto create the amplification product. This technique can be combined witha number of different selective mass label release methodologies toidentify sequences.

[0096]FIG. 4C illustrates two different methods for creatingmass-labeled polynucleotide probes by either transcription (A) or PCRamplification (B). Use of RNA transcription to synthesize mass-probes islimited to sequence regions that are downstream from a promoter sequence(labeled P). Typical synthetic procedures would utilize RNA polymeraseand ribonucleoside triphosphates, including mass-labeled versions thatmay carry one or more mass labels. Shown in (A) is a transcriptionvector carrying a transcription promoter and a clone insert sequence tobe transcribed downstream. The vector also carries one or morerestriction sites (labeled R) that may optionally be cut to control thelength of transcripts. Virtually any amplification technique may be usedto create mass-labeled probes including PCR, as is shown in (B). PCRamplification requires the use of two opposing primers to enableexponential amplification of the sequence located between them. One ormore mass labels may be placed on one or both of the primers oroptionally incorporated through the use of mass-labeled nucleosidetriphosphates.

[0097]FIG. 5A and FIG. 5B illustrate schemes for detecting mutationsusing mismatch specific techniques with enzymatically synthesizedmass-labeled probes. Generally the methodology requires the crosshybridization of normal and mutant or polymorphic nucleic acid to form adouble-stranded product containing a mismatch; enzymatic or chemicalcleavage at the site of the mismatch; and cleavage induced digestion ofthe probe to release one or more mass labels. In the example shown inFIG. 5A and continued in FIG. 5B, a double-stranded mass-labeled nucleicacid probe is synthesized using PCR (A), the 3′ ends of the product areblocked from exonuclease digestion (B), the PCR probe is hybridized tomutation carrying DNA (C) which leads to the formation of a base-pairmismatch, the mismatches are cleaved (D), the cleaved products aredigested with a 3′ to 5′ exonuclease (E), the mass labels are released(F) and analyzed by mass spectrometry (G). Examples of 3′ exonucleaseblocking groups include nucleotide mimics incorporated near the 3′ end,such as nucleotides contains boranophosphates or phosphorothioates, orthe use of 3′ overhangs created during nested-set PCR or by templateindependent extension by terminal transferase in combination with adouble-strand-specific 3′ to 5′ exonuclease, such as exonuclease III,that does not recognize or digest 3′ overhangs. Examples of mismatchspecific cleavage agents for use in (D) include the chemical OSO₄,KMnO₄, and HONH₂, and enzymes, such as mutHLS, T4 endonuclease VII, mutYDNA glycosylase, thymine mismatch DNA glycosylase, or endonuclease V.Methods using RNA or RNA/DNA hybrids are also possible.

[0098]FIG. 6A, FIG. 6B and FIG. 6C illustrate schemes for the synthesisof peptide-linked nucleoside triphosphates (FIG. 6A), an oligonucleotidewith a linker molecule that contains a release group, a disulfide, and aterminal amino-modification for coupling a peptide of some other masslabel component to the end (FIG. 6B), and a scheme for the synthesis ofa peptide-linked nucleoside phosphoramidite (FIG. 6C).

[0099]FIG. 7A and FIG. 7B. show the mass spectra of the unconjugatedoligonucleotide (FIG. 7A) and the oligonucleotide-peptide conjugate(FIG. 7B) of Example 1 D. The spectrum of FIG. 7A contains in additionto the signal for the desired oligonucleotide at m/z 7052, signalsshowing the presence of two significant synthesis failure thatcorrespond to one base and three bases less, and also signals of doublycharged ions for each of these. The spectrum of FIG. 7B shows that thepurified conjugate is of similar purity to the starting oligonucleotide.

[0100]FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D show the mass spectra of ahybridized, mass-labeled probe and target in a buffer after ExonucleaseIII digestion (FIG. 8A), a hybridized, mass-labeled probe and targetincubated with no Exonuclease III (FIG. 8B), of a mass-labeled probe inbuffer incubated with Exonuclease III (FIG. 8C), of a mass-labeled probeincubated with Exonuclease III buffer in the presence of anon-complementary 36-mer target (FIG. 8D). As shown in these FIGS., themass label is released only in the presence of the exonuclease and acomplementary target strand.

[0101]FIG. 9A, FIG. 9B and FIG. 9C compare solid support grid assaysusing a radioactively-labeled probe (FIG. 9A), fluorescently-labeledprobes (FIG. 9B) and mass-labeled probes (FIG. 9C).

[0102]FIG. 9A describes the classical approach to probing nucleic acidsamples arrayed on a spaced grid. Commonly nucleic acid samplesrepresenting mRNA isolates, cDNA clones, genomic clones are arrayed on anylon membrane or filter grid (A). Following a photocrosslinking processto covalently attach the samples to the membrane, a radioactive probe(B) (labeled A), in solution, is added and incubated with the grid (C).The probe hybridizes to positions in the grid where the nucleic acidsamples contain a length of sequence complementary to the probe. Afterwash step the grid is exposed to X-ray film and the hybridizationpositions are identified (indicated by the A positions in the grid) (D).

[0103]FIG. 9B illustrates the extension of the process in FIG. 9A, tothe use of fluorescently-labeled probes (B). Because of the differentemission spectra of different fluorescent labels it is possible tomultiplex a small number, e.g. 4 (labeled A, B, C, D), of differentlylabeled fluorescent probes and cross hybridize them to the grid (C). Inthe case where fluorescence is used, the grid may be composed on a glassplate, rather than a filter or membrane, to enable fluorescence scanningtechniques.

[0104]FIG. 9C illustrates the use of mass-labeled probes (B) (labeledA-S) for hybridization against a gridded array of nucleic acid samples.Either single or combinatorial labeling techniques may be used to createa few to millions of different probes, all simultaneously hybridizedagainst the array. The grid (D), which may be a nylon membrane or someother conducive material may be scanned directly in the massspectrometer following hybridization, wash, mass-label release, andmatrix addition steps (C). Scanning each position of the grid in themass spectrometer reveals one of the many possible mass-label signaturesassociated with each unique probe. Typical examples of assays that woulduse this technology include the use of known gene-specific probesagainst gridded cDNA clones, mRNA, cDNA or amplified cDNA pools. Genomicprobes, both known or unknown against gridded genomic clones. mRNA,cDNA, amplified cDNA against known gridded genes.

[0105]FIG. 10A and FIG. 10B compare library expression analysis using afluorescence based system (FIG. 10A) and a mass-labeled system (FIG.10B). Fluorescence labeling of pairs of cDNA pools derived from mRNA isused to cross compare the gene expression patterns between two differentbiological samples.

[0106] in FIG. 10A, one cDNA pool is labeled with fluorescent tag Awhile the other pool is labeled with fluorescent tag B (A). These poolshave their concentrations normalized and are mixed (B). The mixture ofthe pools is then hybridized against a gridded, reference array of knowngenes, typically arrayed as cDNA clones. Following hybridization thearray is scanning fluorimetrically and the ratio of the two tags ismeasured for each location. For a given location if tag A is twice theintensity of tag B, it is determined that the gene, which is gridded tothat location, is expressed as mRNA at twice the concentration forsample A than for sample B.

[0107]FIG. 10B, expands the concept of competitively hybridizing cDNApools beyond the 2 pool level. The use of releasable mass labels providethe means for the preparation of many more pools (A) (labeled A-H),cross-competitive hybridization (B), and detection (C) of many morepools of expressed message all simultaneously.

[0108]FIG. 11 illustrates the basic principal of release of a mass labelfrom a nucleic acid probe for analysis by mass spectrometry. The masslabel, M1, is released either chemically or enzymatically (A) anddetected by mass spectrometry (B).

[0109]FIG. 12 illustrates selective release of mass labels followinghybridization of a nucleic acid probe to a target DNA sequence.Mass-labeled nucleic acid probes (A), that may contain more than onelabel (as shown), and having different masses of mass label (not shown),are hybridized to a complementary nucleic acid target (B) to form adouble-stranded complex (C). This complex is recognized by adouble-strand-specific exonuclease and the probe is digested (D),releasing mass labels from the probe (E). For processive exonucleasesthe process will continue (F) until the entire probe is digested (G).The digestion is then analyzed by mass spectrometry and the releasedmass labels are detected (H). Mass labels comprise at least onenucleotide when digested by an exonuclease.

[0110]FIG. 13 illustrates the separation of peptides A-G by MALDI massspectrometry where A is angiotensin I, B is substance P, C isCGYGPKKKRKVGG (SEQ ID NO:2), D is TCVEWLRRYLKN (SEQ ID NO:7), E isCSRARKQAASIKVSADR (SEQ ID NO:8), F is exidized A-chain insulin and G ismelittin.

[0111]FIG. 14 illustrates a schematic representation of a process bywhich a series of gene-specific mass-labeled nucleic acid probes areused to detect and quantify the amount of different targeted mRNAswithin a given sample. A starting pool of nucleic acid (A) that is themRNA, cDNA copy of the mRNA, or some amplified multiplex of nucleic acidderived from the mRNA, is mixed with a set of message-specificmass-labeled nucleic acid probes (B) (probes with different mass labelslabeled A-S). The mixture is allowed to hybridize (C) wherein probesthat find complementary messages in the pool form double-strandedcomplexes, wherein the concentrations of the gene-specificdouble-stranded complexes is proportional to the levels of mRNA presentin the starting material. Following the formation of double-strandedcomplexes, the mixture is treated with a double-strand-specificnuclease, e.g. exonuclease III treatment, selectively releasing masslabels from probes that had hybridized (D). The released mass labels(labeled A-s) are then analyzed by mass spectrometry (E), wherein thequantity of each mass label detected is proportional to the levels ofmRNA present in the starting material. The selective release step mayoptionally use double-stranded chemical release probes as well as solidphase capture methods to differentiate double-stranded probes fromunhybridized single-stranded probes.

[0112]FIG. 15A and FIG. 15B shows two mass spectra. For FIG. 15A, anrtPCR™ reaction was performed using a pair of mass-labeled primerstargeted at the mRNA for ribosomal protein L7. Following the PCR™, thereaction mix was treated with the double-strand-specific exonuclease T7gene 6 exonuclease. Only when a double-stranded PCR™ product is formeddoes the exonuclease digest the product and release the two mass labels,as indicated by two peaks in the spectrum. In FIG. 15B, a control wasperformed where a single-stranded, mass-labeled primer was incubatedwith T7 gene 7 exonuclease. No digestion occurred.

[0113]FIG. 16 illustrates the release of a series of seven differentmass-labeled probes which were hybridized to seven different cDNAplasmids and then treated with exonuclease III. An aliquot of thedouble-strand-specific digestion was taken and analyzed by massspectrometry. The mass spectrum is shown with the peaks corresponding toeach mass label signal labeled A-G.

[0114]FIG. 17A and FIG. 17B shows two mass spectra from a SNP analysisusing a mass-labeled primer and a biotinylated dideoxynucleosidetriphosphate. In FIG. 17A a complementary match is made between thepolymorphic base on the template and the biotinylated dideoxynucleosidetriphosphate. The mass-labeled primer has been extended and thereforebiotinylated, which allows it to be captured to a streptavidin-coatedsurface, washed and subsequently cleaved from the surface. FIG. 17Bshows a mass spectrum from a reaction in which the base at thepolymorphic site is not a complementary match to the biotinylateddideoxynucleoside triphosphate present in the reaction. No extension ofthe primer occurred as evidenced by the absence of a mass spectrometricsignal for the primer mass label. The unextended primer is not capturedon the streptavidin-coated surface and is removed in the subsequentwashes.

[0115]FIG. 18 shows a mass spectrum from a multiplex SNP analysis inwhich three differently mass-labeled primers for three differentpolymorphic sites are all simultaneously extended with a biotinylateddideoxynucleoside triphosphate. The three extended primers are allcapable of being captured on a streptavidin-coated surface, washed toremove unextended primers and then cleaved from the surface.

[0116]FIG. 19A and FIG. 19B shows two mass spectra from a SNP analysisin which the extension is carried out a few bases past the polymorphicsite and for which biotin is incorporated through a biotinylateddeoxynucleoside triphosphate. The mixture of triphosphates in thereactions consists of deoxy-ATP, biotinylated-deoxy-CTP, anddideoxy-TTP.

[0117] In FIG. 19A the spectrum is from a reaction in which thepolymorphic site on the template, located one base past the 3′-end ofthe primer, is a T. Since the polymorphic site is a complementary matchto one of the deoxynucleoside triphosphates in the reaction, the primeris extended past the polymorphic site, and subsequently incorporates abiotinylated-dCTP before terminating chain extension with thedideoxynucleoside triphosphate.

[0118] The reaction whose spectrum is shown in FIG. 19B is one in whichthe polymorphic site on the template is A. Therefore a dideoxy-TTP isincorporated at the first base past the primer, and chain extension isterminated prior to incorporation of the biotinylated-dCTP, whichresults in a lack of signal in the mass spectrum.

[0119]FIG. 20A and FIG. 20B show two mass spectra from primer extensionanalyses in which a mixture of three primers, differing only in their3′-end-bases and each containing unique mass labels, is extended withbiotinylated dideoxynucleoside triphosphate. In FIG. 20A the massspectrum shows signal predominantly for the primer whose 3′-end base(primer A) is a perfect match for the template used in the reaction. Thespectrum in FIG. 20B is from a reaction in which the template is changedfrom the reaction in FIG. 20A in such a way that the 3′-end base matchesto a different primer and gives predominantly signal from extension ofprimer E.

[0120]FIG. 21A and FIG. 21B show two mass spectra comparing the chemicalcleavage rates for double-stranded versus single-stranded DNA. Acleavable oligonucleotide containing a 5′-S-P bond is cleavable byAgNO3. Two cleavage reactions are run. In the first reaction thecleavable oligonucleotide is hybridized to a complementaryoligonucleotide to make it double-stranded prior to adding cleavagereagent. The second reaction is performed on single-strandedoligonucleotide. The mass spectrum in FIG. 21A shows the products fromcleavage of double-stranded DNA. The cleavage products are expected atmasses of 6560 Da and 1470 Da, while the uncleaved oligonucleotide isseen at 8012 Da. The spectrum of FIG. 21A indicates that only about 5%cleavage has occurred. The spectrum in FIG. 21B, which is from cleavageof single-stranded oligonucleotide demonstrates that under the sameconditions, cleavage is about 90% complete.

[0121]FIG. 22A and FIG. 22B show two mass spectra from a probe assay ofa gene-specific RNA transcript. Two exonuclease III digestions reactionsare run. In both reactions a mixture of two probes is present and thetemplate consists of either RNA transcript or the DNA PCR-producttemplate from which the RNA is transcribed. Only one of the probes iscomplementary to the RNA transcript the other probe is complementary tothe opposite strand. Therefore is mass label signal is obtained from theDNA PCR product, signals for both probes are seen, while if the signalis obtained from RNA transcript, only one signal is seen.

[0122] in FIG. 22A the mass spectrum shows the resulting released masslabel for the reaction in which RNA transcript is present. Since onlyone signal is seen, the signal must come from digestion of the probehybridized to the RNA transcript. The second reaction contains a100-fold greater amount of DNA PCR product than is present in the firstreaction, and no RNA transcript.

[0123]FIG. 22B shows the mass spectrum resulting from the secondreaction. The presence of signals from both probes confirms the factthat the signal in FIG. 22A comes from RNA-hybridized probe.

[0124]FIG. 23A, FIG. 23B, FIG. 23C, and FIG. 23D show a set of four massspectra which compare the analyte selectivity of two different matricesfor MALDI. The samples used for the comparison are equimolar mixtures ofa nucleotidylated peptide and an oligonucleotide obtained by a selectivechemical cleavage of an oligonucleotide-peptide conjugate. FIGS. 23A and23B compare spectra of the same sample obtained with2,5-dihydroxybenzoic acid matrix (FIG. 23A) and with 3-HPA matrix (FIG.23B). The peptide signal predominates in FIG. 23A while theoligonucleotide predominates in spectrum FIG. 23B due to differingdesorption selectivities or efficiencies of the matrices for the peptideand the oligopeptide. The spectra in FIG. 23C and 23B make the samecomparison with a different sample showing that the ionizationselectivity is general.

[0125]FIG. 23 illustrates the use of a double-stranded, mass-labelednucleic acid probe for detecting and quantifying the presence of anucleic acid target sequence. Contained within the double-stranded probeis a chemical cleavage group that, under proper conditions, only cleaveswhen the nucleic acid probe is single-stranded. Examples of chemicalcleavage groups that demonstrate enhanced cleavage rates when singlestranded include chemically labile nucleic acid backbone modificationssuch as 5′-(S)-phosphorothioate, 3′-(S)-phosphorothioate,5′-(N)-phosphoramidate, 3′-(N)-phosphoramidate, and ribose. Probing of anucleic acid target sequence involves combining the double-strandedprobe (A) with the single-stranded target (B) and allowing them todenature and anneal under equilibrium conditions (C). The probe strandcontaining the mass label and single-strand-specific release group(labeled Re) is homologous to the target nucleic acid; the complementarystrand is also complementary to the target. The other products of thisequilibrium event are the mass-labeled, cleavable strand insingle-stranded form (D), and the complementary strand annealed to thetarget (E). The amount of complementary strand released from themass-labeled strand and annealed to the target is proportional to theconcentration of the target nucleic acid. Following the annealingprocess the probes are treated with a single-strand-specific chemicalcleaving agent (F) yielding cleaved single-stranded probe (G) anddetected and quantitated by mass spectrometry (H). As with othermass-labeled probes described here, the mass label may be wholely oronly partially contained within the nucleic acid probe or reactive groupand may include the use of nucleic acid mimics.

[0126]FIG. 25 illustrates the use of mass-labeled substrates inenzyme-linked affinity assays. Specifically illustrated are the caseswhere the target molecule (labeled T) is a protein (A) and a nucleicacid (B). In illustration (A), an antibody (labeled Ab) is used torecognize the solid-phase bound target. The antibody is conjugated tothe enzyme (labeled E) used to produce signal. In this particularaffinity assay, the enzyme recognizes a mass-label substrate (labeledMX) and converts it to product which in this example is a cleavage eventto form two products (labeled M and X) which are then analyzed by massspectrometry. Regarding the mass label substrates, the primaryrequirement is that the enzyme modify the mass of the substrate when itis converted to product by either adding or removing chemical moietiesfrom the substrate. In illustration (B), the antibody has been replacedby a nucleic acid probe that is then conjugated to the signal producingenzyme. The assay is extremely generalizable and one skilled in the artwould be able to identify a variety of combinations of probe and target,as well as enzymes and mass-label substrates that may be used.

[0127]FIG. 26 illustrates two examples of mass-label substrates for usein enzyme-linked affinity assays. Specifically illustrated are twoexamples, (A) a double-stranded oligonucleotide containing a restrictionendonuclease site (labeled R), and (B) a polypeptide containing aspecific proteolytic linkage. In both examples it is possible to developa repertoire of enzymes and mass-label substrates, since a variety ofrestriction endonucleases and proteases exist that exhibit eithersequence-specific or monomer-specific cleavage activity. Use of theseclasses of enzymes allow a plurality of affinity assays to take placesimultaneous within the same reaction vial. All producingmass-differentiable mass-label products. As with other mass-labeledprobes described here, the mass label may be wholely or only partiallycontained within the nucleic acid or polypeptide substrate and mayinclude the use of nucleic acid mimics or non-natural amino acids.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0128] The present invention is directed to the composition and use ofreleasable, nonvolatile mass labels for chemical analysis. The masslabels will be detectable by mass spectrometry. The present inventionalso describes novel methods utilizing mass labels of any form. The termnonvolatile as used herein refers to a molecule which when present inits pure, neat form and heated, does not sublimate intact to anysignificant extent. Also included in the definition of nonvolatilecompounds are compounds which when present in their pure, neat formcannot be practically analyzed by mass spectrometry when conventionalgas chromatography is employed in the sampling process. An advantage ofusing nonvolatile mass labels versus volatile mass labels is that thesample mixtures are thereby easily physically stable after release. Themass labels described may be attached to a probe molecule that canspecifically interact with the intended target. In some cases, a specialrelease group may be included to chemically link the mass label to theprobe.

[0129] It is also possible to use mass labels which have negligiblevapor pressure at room temperature but can be considered volatile by theabove definition. In the present work, the novel mass labels releasedfrom the probe molecule evaporate insignificantly if at all at roomtemperature and are not efficient electrophores. Molecules belonging tothis category are termed involatile mass labels.

[0130] The compounds of the present invention are useful for detecting awide variety of biomolecular interactions. Representative examplesinclude identification of gene sequences, identification of non-codingnucleotide sequences, identification of mutations within a gene orprotein sequence, detection of metals, detection of toxins, detection ofreceptors on an organism or a cell, characterization of antibody-antigeninteractions, enzyme-substrate interactions and characterization ofligand interactions.

[0131] A. Mass Labels

[0132] Mass label is a term that can be used synonymously with tag orsignal. Examples of the types of mass labels for the present inventioninclude a repertoire of compounds, preferably ones that share similarmass spectrometric desorption properties and have similar or identicalcoupling chemistries in order to streamline synthesis of multiple masslabel variants. A mass label of the present invention is detectable bymass spectrometry. Representative types of mass spectrometric techniquesinclude matrix-assisted laser desorption ionization, directlaser-desorption, electrospray ionization, secondary neutral, andsecondary ion mass spectrometry, with laser-desorption ionization beingpreferred. The dynamic range of mass spectral measurements can generallybe extended by use of a logarithmic amplifier and/or variableattenuation in the processing and analysis of the signal. An example ofa peptide mixture separated by mass spectrometry is shown in FIG. 13.

[0133] Mass labels may include a vast array of different types ofcompounds including biopolymers and synthetic polymers. Representativebiological monomer units that may be used as mass labels, either singlyor in polymeric form, include amino acids, non-natural amino acids,nucleic acids, saccharides, carbohydrates, peptide mimics and nucleicacid mimics. Preferred amino acids include those with simple aliphaticside chains (e.g., glycine, alanine, valine, leucine and isoleucine),amino acids with aromatic side chains (e.g., phenylalanine, tryptophan,tyrosine, and histidine), amino acids with oxygen and sulfur containingside chains (e.g., serine, threonine, methionine and cysteine), aminoacids with side chains containing carboxylic or amide groups (e.g.,aspartic acid, glutamic acid, asparagine and glutamine), and amino acidswith side chains containing strongly basic groups (e.g., lysine andarginine), and proline. Derivatives of the above described amino acidsare also contemplated as monomer units. An amino acid derivative as usedherein is any compound that contains within its structure the basicamino acid core of an a amino-substituted carboxylic acid, withrepresentative examples including but not limited to azaserine,fluoroalanine, GABA, ornithine, norleucine and cycloserine. Peptidesderived from the above described amino acids can also be used as monomerunits. Representative examples include both naturally occurring andsynthetic peptides with molecular weight above about 500 Daltons, withpeptides from about 500-5000 Daltons being preferred. Representativeexamples of saccharides include ribose, arabinose, xylose, glucose,galactose and other sugar derivatives composed of chains from 2-7carbons. Representative polysaccharides include combinations of thesaccharide units listed above linked via a glycosidic bond. The sequenceof the polymeric units within any one mass label is not critical; thetotal mass is the key feature of the label.

[0134] The monomer units according to the present invention also may becomposed of nucleobase compounds. As used herein, the term nucleobaserefers to any moiety that includes within its structure a purine, apyrimidine, a nucleic acid, nucleoside, nucleotide or derivative of anyof these, such as a protected nucleobase, purine analog, pyrimidineanalog, folinic acid analog, methyl phosphonate derivatives,phosphotriester derivatives, borano phosphate derivatives orphosphorothioate derivatives.

[0135] Mass labels according to the present invention may also includeany organic or inorganic polymer that has a defined mass value, remainswater soluble during bioassays and is detectable by mass spectrometry.Representative synthetic monomer units that may be used as mass units inpolymeric form include polyethylene glycols, polyvinyl phenols,polymethyl methacrylates, polypropylene glycol, polypyroles, andderivatives thereof. A wide variety of polymers would be readilyavailable to one of skill in the art based on references such as Allcocket al. (1981) which describes the properties of many additional polymerscontemplated for use in the present invention. The polymers may becomposed of a single type of monomer unit or combinations of monomerunits to create a mixed polymer. The sequence of the polymeric unitswithin any one mass label is not critical; the total mass is the keyfeature of the label.

[0136] For nonvolatile mass labels having mass below about 500 Da,usually significant ionic character is required; representative examplesinclude polyethylene glycol oligomers of quaternary ammonium salts(e.g., R—(O—CH₂—CH₂)n-N(CH₃)₃ ⁺.Cl⁻) and polyethylene glycol oligomersof carboxylic acids and salts (e.g., R—(O—CH₂—CH₂)_(n)—CO₂ ⁻.Na⁺).

[0137] Examples of involatile mass labels typically include smalloligomers of polyethylene glycol and small peptides (natural ormodified) less than about 500 Da in molecular weight. In theseinstances, as for all of the cases considered herein, mass analysis isnot by electron attachment.

[0138] Mass labels of the present invention may also include a varietyof nonvolatile and involatile organic compounds which are nonpolymeric.Representative examples of nonvolatile organic compounds include hemegroups, dyes, organometallic compounds, steroids, fullerenes, retinoids,carotenoids and polyaromatic hydrocarbons.

[0139] In addition to the polymer or mixed polymer mass labelsdescribed, mass-labels or the present invention also include mixed masslabels containing a mass-variable polymeric component and a nonpolymericmass static component. A representative example includes a set of masslabels with a polymeric component where the number of repeat unitswithin the set is a range from about 10 to 100, and on each polymer is acompound with a fixed large mass. In a preferred embodiment, the masslabels within a set all contain the same mass static component. In thispreferred set of compounds only the length of the polymer is changed toprovide a set of mass labels with incremental increases in mass and arelatively uniform signal between mass labels. These compounds provide ameans for using mass labels with desirable spectral properties but arenot available in a large repertoire of different masses.

[0140] It is preferable when using multiple mass labels on a probe, toavoid signal overlap. In addition to presenting a large, primary signalfor a mass label with a single charge, there is also the potential formultiply charged versions of a mass label to present a signal as well asdimerized versions of a mass label. The presence of multiple signals fora single mass label can potentially overlap with and obscure the signalfor the primary peak of a second mass label. Thus typically the range ofmass labels used for a given analysis may have a mass range where nomultiply charged or dimer species can interfere with the detection ofall mass labels, for example, the mass labels may have a range of masseswherein the smallest mass-label is more than half the mass of thelargest mass label.

[0141] B. Reactive Groups

[0142] The mass label is typically attached to a reactive group. Thereactive groups of the present invention may be any biomolecule capableof specific molecular recognition. In particular, the reactive group mayform a specific interaction with the target molecule. This interactionmay be noncovalent, for example, hybridization of an oligonucleotide toa DNA target, or covalent such as crosslinking. Representative reactivegroups of the present invention include polypeptides, antibodies,enzymes, polynucleic acids, lipids, steroids, carbohydrates, antibioticsand compounds such as neocarzinostatin which have a preference forcertain DNA sequences, with polynucleic acids preferred andoligonucleotides being more preferred. Representative steroid hormonesinclude estrogens, progestins and androgens.

[0143] Representative reactive group-target molecule interactionsinclude oligonucleotide-oligonucleotide hybridization,polynucleotide-polynucleotide interactions, enzyme-substrate orsubstrate analog/intermediate interactions, polypeptide-nucleic acidinteractions, protein-ligand interactions, receptor-ligand interactions,lipid-lipid interactions, carbohydrate-carbohydrate interactions,polypeptide-metal interactions, nucleic acid-metal interactions orantigen-antibody interactions.

[0144] In certain embodiments the probe may be a syntheticoligonucleotide or enzymatically synthesized oligonucleotide that may bea DNA molecule, an RNA molecule, or some variant of those molecules,such as a peptide nucleic acid. The oligonucleotide will typically beable to selectively bind a substantially complementary sequence. As usedherein a substantially complementary sequence is one in which thenucleotides generally base pair with the complementary nucleotide and inwhich there are very few base pair mismatches. The polynucleotide may berelatively small, such as a 10-mer, or larger, such as a kilobase insertin a plasmid or a kilobase amplified nucleic acid (“amplicon”) or a longRNA transcript. The polynucleotide can be bigger, smaller or the samesize as the target. The probe is distinguished from the target by thefact that the probe contains a mass label.

[0145] Representative examples of a covalent interaction between areactive group and a target include proteins as reactive groupsactivated with crosslinkers to form conjugates with the target molecule,such as antibody-antigen interactions, enzyme-substrate interactions,receptor-ligand interactions, receptor-membrane interactions or aprotein-nucleic acid interaction. Representative crosslinking reagentsinclude chemically activated crosslinkers such as EDC or MBS andphotoreactive crosslinkers such as SADP or PNP-DTP.

[0146] C. Methods for Releasing the Mass Label

[0147] In some embodiments, it may be important to release the masslabel from all or most of the reactive group prior to spectrometricanalysis, as represented in FIG. 11 for a mass-labeled nucleic acidprobe. For this reason, a release group is desirable. A number of meansmay effectuate the release, including a labile chemical linkage betweenthe mass label and the reactive group. A labile chemical linkage as usedherein is any moiety which upon treatment with a second chemical agent,light, enzyme or heat will cleave the moiety and release the mass label.These linkages may include chemically cleavable groups incorporatedwithin the phosphate backbone linkage (e.g. replacement of phosphatewith a phosphoramidate) or as a substituent on or replacement of one ofthe bases or sugars of the oligonucleotide primer (e.g., a modified baseor sugar, such as a more labile glycosidic linkage). Such chemicallycleavable groups would be apparent to one of skill in the art in lightof the present disclosure and include, for example, dialkoxysilane,3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate,3′-(N)-phosphoroamidate, 5′-(N)-phosphoroamidate, and ribose. It hasalso been found experimentally that such groups cleave much more rapidlywhen the probe is in single-stranded form than when hybridized to acomplementary strand. An example of this kinetic selectivity ispresented in Example 9. The chemically cleavable site should generallybe stable under the amplification, hybridization and washing conditionsto be employed. Other examples of labile chemical, linkers consist ofgroups cleavable by oxidation such as dialkyl tartrate, base cleavablegroups such as bis[2(alkoxy-carbonyloxy)ethyl]sulfone, silyl ethers andketals which will cleave upon treatment with fluoride ion or acid,ortho-nitrobenzyl ethers which will cleave upon irradiation with light,and groups cleavable by reduction such as dialkyl disulfides.

[0148] A preferred labile chemical linkage includes a disulfide bondwhich upon treatment with a sulfhydryl reagent, such as2-mercaptoethanol, reduces the disulfide bond into two —SH groups. Formass labels that are chemically cleaved from probes, it may bepreferable to remove or wash away any unincorporated reactive groupmonomers so that they are not visualized in the mass spectrometer.

[0149] In other embodiments of the invention, however, no additionallinkage group will be needed, as the release group may be containedwithin the reactive group. Released mass labels therefore, may containnone, a portion, or the whole of the reactive group still attached tothe specific mass label. Representative examples of release groupscontained within a reactive group include the endogenous peptidelinkages between amino acids in a polypeptide and the endogenousphosphodiester bond linkages between bases in a polynucleotide. When thereactive group is a polynucleotide, the mass label may be releasedduring enzymatic (nuclease) digestion of the probe nucleotide backbone,or an acid-induced digestion of the probe nucleotide backbone. Theseendogenous linkages may also be modified to target a specific sequencewithin the reactive group. Examples include modified phosphodiesterbonds such as phosphorothioates, phosphoramidates and dialkylsilylketals. Nucleotide sequences may also be introduced for recognition byan endonuclease (restriction enzyme) such as Type II or Type IISrestriction endonucleases. In certain embodiments a phosphodiester bondwill be the release group as recognized by an exonuclease enzyme.Temperature labile release is also contemplated. Representative examplesinclude thermal melting of a hybridized oligonucleotide from a DNAtarget or temperature dependent denaturation of a protein to release abound molecule.

[0150] Specific peptide linkages may also be introduced within apolypeptide reactive group. Examples include peptide linkages which arespecifically cleaved by chemicals such as a methionine recognized byCNBr, or tryptophan which can be cleaved by either lodosobenzoic acid orBNPS-skatole. Peptide linkages may also be introduced for recognition byan enzyme such as trypsin.

[0151] A further example of endogenous bonds as release groups includechemical or enzymatic cleavage at a glycosidic bond. One skilled in theart would recognize that a wide variety of release approaches would bewithin the scope of the present invention.

[0152] D. Selective Release of Mass Labels

[0153] In some of the embodiments described herein, involving the use ofone or more different nucleic acid probes, use of mass-labeled nucleicacid probes may depend on the selective release of certain mass-labelscorrelating to the occurrence of a particular event. For instance,release of a mass-label may indicate that a hybridization event hasoccurred between a particular mass-labeled nucleic acid probe and anucleic acid target sequence. An approach to selective release caninvolve targeted nuclease digestion of only hybridized probes existingin a double-stranded form as shown in FIG. 12. A number of nucleases,for example restriction endonucleases and DNase 1, only digestdouble-stranded nucleic acids. Consequently treatment with such enzymeswill only release mass-labels from nucleic acid probes that havesuccessfully hybridized to a target sequence. As an alternative, anuclease that only recognizes a nucleic acid sequence present insingle-stranded form, including S1 nuclease, could be used to yieldsignal and identity data for probes that do not undergo hybridization.

[0154] The use of a hybridization probe of at least about 10-14nucleotides in length allows the formation of a duplex molecule that isboth stable and selective. Molecules having contiguous complementarysequences over stretches greater than 10 bases in length may be employedto increase the stability and selectivity of the hybrid. One maygenerally prefer to design nucleic acid molecules having complementarystretches of about 15 to about 20 contiguous nucleotides, or even longerwhere desired. For example, one may prefer to design nucleic acidmolecules of about 25, about 30, about 35, about 40, about 45, or about50 contiguous nucleotides and so on. In this context, the term “about”indicates that the nucleic acid molecule may vary from the stated lengthby from 1 to 4 nucleotides. For example, “about 25” may be understood toinclude 21, 22, 23 and 24; “about 30” may be understood to include 26,27, 28 and 29; “about 35 may be understood to include 31, 32, 33 and 34;and so on.

[0155] Hybridization probes may be selected from any portion of a targetsequence. The choice of probe and primer sequences may be governed byvarious factors, such as, by way of exemplification and not limitation,one may employ primers from regions near the termini of the totalsequence, or from the ends of the functional domain-encoding sequencesor one may employ probes corresponding to the entire DNA. Probes may bedesigned to identify homologous genes between species including human orone may employ wild-type and mutant probes or primers with sequencesdesigned to identify human or other non-human subjects that carry acertain mutation and thus may be susceptible to disease or apharmaceutical agent.

[0156] Variable parameters for hybridization include temperature, time,salt concentration and formamide concentration. Hybridization isunderstood to mean the formation of stable, anti-parallel duplexmolecules based on the specific hydrogen bonding of complementarynucleotide bases of the nucleic acid molecules

[0157] The tendency for two complementary strands of nucleic acid insolution to anneal or hybridize by forming hydrogen bonds between theircomplementary bases, is critically dependent on the concentration ofmonovalent or divalent cations in the solution. Sodium (Na⁺), has beenthe cation of choice for determining the effects of salt concentrationon the stability of duplex nucleic acids. Above the threshold Na⁺concentration, two complementary single strands (either DNA or RNA) ofnucleic acid will hydrogen bond through interaction of the bases in eachstrand, to form a double-stranded molecule of DNA, RNA, or even aDNA-RNA heteroduplex. Complementary bases are adenosine (A) andthymidine (T) (in DNA), or adenosine and uridine (U) (in RNA), andcytosine (C) and guanine (G) in both DNA and RNA. Two hydrogen bonds areformed between paired A and T or A and U residues, while C-G basepairing results in the formation of three hydrogen bonds. The G-C basepair is therefore a stronger interaction than the A-U or A-T base pair.In general, hydrogen bonding (leading to duplex formation) does notoccur between non-complementary bases. The ability of two single strandsto form a stable double-stranded duplex depends on the sequence of basesin each strand being complementary to the other, such that when thestrands are aligned in an antiparallel orientation, sequentialjuxtaposed bases are able to form hydrogen bonds. Although hydrogenbonding between any two complementary bases provides only a weak bindingenergy, the cumulative binding energy between many sequential pairedbases provides sufficient attractive forces to hold the strands togetherin a stable duplex. Cations enhance the tendency for complementarystrands to form hydrogen bonds, by masking the negative charges of thephosphate groups in the phosphodiester linkages which form the“backbone” of the nucleic acid strands. At low concentrations ofpositively charged ions, repulsive forces between negatively chargedstrands favor their single-stranded or denatured conformation; as cationconcentration is raised, the negative charges are masked, complementarybases pair through hydrogen bonding, and a duplex nucleic acid moleculeis formed. In a duplex containing a mismatched (non-complementary) basepair, the single unpaired position in the two otherwise complementarystrands provides the target for the single-strand specific RNase in theRNase protection assay.

[0158] Other parameters besides cation concentration affect the tendencyof complementary strands to exist in the alternative double-stranded orsingle-stranded conformations. Temperature is a critical variable; asthe temperature of a solution of duplex nucleic acid molecules israised, hydrogen bonds are broken first in A-U rich regions and finallyin G-C rich regions, until above a critical temperature, thecomplementary strands come apart. The composition of the two strands,i.e., their % GC content, determines the critical temperature for duplexdenaturation at a given ionic strength. As a corollary, the % GC alsodetermines the threshold concentration of Na⁺ needed to maintain duplexstability at a given temperature. Stability of duplex nucleic acidmolecules in solution is also affected by the nature of the solvent. Forexample, duplexes are much less stable in formamide (which destabilizeshydrogen bonds) than in aqueous solution, a fact exploited by molecularbiologists to achieve nucleic acid hybridization at lower temperaturesthan would otherwise be required.

[0159] Equations have been derived to relate duplex formation to themajor variables of temperature, salt concentration, nucleic acid strandlength and composition, and formamide concentration. Eg:

Tm=81.5−16.6(log[Na⁺])+0.41(%GC)−600/N  1.

[0160] (Tm=temperature for duplex to half denature; N=chain length

Tm=81.5−16.6(log[Na⁺]+0.41(%GC)−0.63(%formamide)−600/N  2.

[0161] One can thus predict whether complementary strands will exist indouble-stranded or single-stranded form under a given set of conditions.If conditions are chosen such that complementary strands form a stableduplex, the duplex will in theory be resistant to the nucleolytic actionof enzymes (DNases and RNases) which are specific for cleavage ofphosphodiester bonds in single-stranded molecules. Many different typesof nucleases exist, which vary widely in their substrate specificities.The RNases commonly used in RNase protection assays are specific forcleavage after particular bases in single-stranded RNA molecules. Belowthe threshold Na⁺ concentration needed t maintain duplex stability, thecomplementary RNA strands denature into single strands, which are thensubstrates for degradation by the RNases. Susceptibility to digestion byRNase A is therefore a functional assay for whether complementarystrands exist as single-stranded or double-stranded molecules.

Hybridization

[0162] Standard annealing or hybridization procedures are described bySambrook et al. (1989). Generally they entail two or more nucleic acids,for example probe and test sample nucleic acids, to be mixed together,denatured and then subjected to conditions in which complementarystrands anneal, or base pair by hydrogen bonding to form double strands.The annealed strands are said to be hybridized. For example, the mixturemay be heated to from about 90° C. to about 95° C. for about threeminutes and then gradually cooled to a lower temperature, 42° C. forexample, for a period of time sufficient to allow hydrogen bonding ofthe complementary strands. The time required for annealing ofcomplementary strands depends on the concentration of each strand andwill vary from a few minutes (for reactions where both probe an testnucleic acids are present at high concentrations), to several hours orovernight for reactions having at least one species present at lowconcentration. It is therefore advantageous to use high concentrationsof probe and test sample nucleic acids, such as may be generated by PCRamplification and/or transcription of PCR amplified sequences.

[0163] Depending on the application envisioned, one may employ varyingconditions of hybridization to achieve varying degrees of selectivity ofthe probe towards the target sequence. For applications requiring highselectivity, one may typically employ relatively stringent conditions toform the hybrids, e.g., relatively low salt and/or high temperatureconditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C. to 70° C. Such selective conditions tolerate little, if any, mismatchbetween the probe and the template or target strand.

[0164] Of course, for some applications, for example, where one desiresto identify mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate protein-encodingsequences from related species, functional equivalents, or the like,less stringent hybridization conditions may typically be employed toform the heteroduplex. In these circumstances, one may employ milderhybridization conditions, such as 0.15M-0.9M salt, at temperaturesranging from 20° C. to 55° C. Cross-hybridizing species can thereby bereadily identified as positively hybridizing signals with respect tocontrol hybridizations. Additionally, conditions may be rendered morestringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions may be readily manipulatedto achieve the desired results.

Release Methods

[0165] The use of nucleases that selectively digest mass-labeled nucleicacid probes hybridized to a target nucleic acid allows for linearamplification of signal. For example, one may employ a nuclease capableof digesting only the nucleic acid probe and not the target, e.g., adouble-strand specific exonuclease to digest a short, linear probe inthe presence of a circular target having no end to enable the initiationof exonuclease digestion. Long linear targets may also be used in caseswhere the exonuclease requires a recessed or blunt double-stranded end.As a probe hybridizes to the target, it is digested, and the digestedfragments release from the target and make room for a second copy of theprobe to hybridize. The second probe is then digested, and, once again,the target is free for the next hybridization. The repeated cycles ofhybridization and digestion leads to a linear amplification of theamount of released mass label in solution, consequently increasing themass spectrometric signal. It is possible to achieve a many hundred-foldamplification of signal using such a system. See Okano and Kambara, 1995(exonuclease III); Copley and Boot, 1992 (lambda exonuclease).

[0166] Nonselective release events may also be employed with the methodsdisclosed herein. For example, nonselective cleavage of a disulfidereleasing group using a chemical agent such as a phosphine or amercaptan may be used.

[0167] In certain embodiments, detection of the desired label may dependon specific partitioning of the population of reactive groups ortargets. Reactive groups that recognize and bind to a particular targetmay, for example, be immobilized to a specific location. For instance, atarget sequence or sequences of nucleic acids may be attached to griddedpositions on a solid support such as a filter, glass, gold or to a beador a group of beads. Mass-labeled oligonucleotides (probes) that do nothybridize to the target sequence may then be separated from probeshybridized to immobilized targets simply by washing the filter or beads.Such approaches may be especially preferred for removal of unhybridizedprobes where a subsequent nonspecific release mechanism is to beemployed. The reverse case may also be employed, in which the labeledprobes are immobilized, and the targets are hybridized to them.

[0168] Methods described herein may involve the use of a nucleic acidamplification event, such as polymerase chain reaction (referred to asPCR™), to link a mass-labeled nucleic acid probe, used specifically as aprimer, to a second primer that is capable of or presently is bound to asolid support. An example of a second primer is one that contains abiotin moiety. Similarly to the embodiment described above, binding ofthe amplification product to the solid phase affords a mechanism to washaway unused primers and then to nonselectively release the remainingmass labels.

[0169] A nucleic acid amplification event, involving the use of one ormore different nucleic acid probes, may also be used to convertmass-labeled nucleic acid probes, used specifically as a primers, fromsingle-stranded form to double-stranded form. This conversion allows theuse of a double-strand-specific nuclease to selectively release onlythose mass labels that were attached to primers involved inamplification events. Unused primers remain single stranded and will notrelease their attached mass labels.

[0170] Other methods described herein as part of the present invention,involving the use of one or more different nucleic acid probes, mayinvolve the modification of a select population of probes followingtheir hybridization to a target which would allow for the partitioningof the probe population. Such methods include double-strand dependentaddition of biotinylated nucleotides or oligonucleotides to the end ofmass-labeled probes using polymerase or ligase, followed by directcapture of the biotinylated probes to a streptavidin modified surface.

[0171] As another option, analysis of mass-labeled nucleic acid probesby MALDI mass spectrometry may be performed using a matrix thatselectively desorbs and efficiently ionizes intact released mass labelsbut not mass labels still coupled to their respective nucleic acidprobes. Nucleic acid molecules often do not desorb well in many matriceswhich are yet effective for the desorption of released mass labels, andthis difference can be accentuated by the presence of impurities such assalts. Mass-labeled nucleic acid probes may typically be analyzed bydirect laser-desorption mass spectrometry without further purificationif, for example, the released mass label(s) are detected much moreefficiently than unreleased labels. The same holds true for other formsof mass spectrometry. Thus, in a preferred embodiment usinglaser-desorption mass spectrometry, physical partitioning of thereleased and unreleased mass labels may not be required. One skilled inthe art in light of the present disclosure can envision the use of avariety of other techniques for selectively partitioning probesinvolving probe-label synthesis, label release, and label mass spectraldetection, in various combinations.

[0172] E. Synthetic Techniques

[0173] Mass labels may be added to the reactive group during synthesis,or the reactive group may be modified after synthesis. For example, themodification of nucleic acid or amino acid building blocks provides aconvenient route for developing generalized methods of mass-labelingreactive groups during synthesis. For example, as the polypeptide orpolynucleic acid is being synthesized, different mass-labelednucleotides or amino acids may be added to the mixture and incorporatedinto the growing polymer. A generalized example of a mass-labelednucleoside triphosphate is depicted in FIG. 1A. One skilled in the artwould in light of the present disclosure envision a variety ofattachment schemes and positions of attachment. Generally, theattachment of a mass label should not substantially inhibit theinteraction between the reactive group and target molecule, such as thehydrogen-bonding of the mass-labeled base and the complementary targetbase, or disrupt the proper folding of a polypeptide to form an activeprotein. Furthermore, in the case of a mass-labeled nucleosidetriphosphate, the label should typically not inhibit polymerization by apolymerase enzyme.

[0174] One synthesis approach of the present invention, involves the useof mass label modified nucleoside triphosphates that are incorporated bya polymerase to produce a mass-labeled polynucleotide. Using thismethod, it is easy to load a nucleic acid probe with many copies of amass label. Polymerase-based methods allow for the inexpensive synthesisof very long probes hundreds to tens of thousands of bases in length byincorporation into an RNA transcript of PCR™ amplicon.

[0175] Where the reactive group is a protein, the mass label may be alength of amino acids forming a peptide attached to either the carboxylor amino terminus of the protein. The composition of the mass label maybe coded directly into the DNA sequence immediately adjacent to thecoding region of the protein that represents the reactive group.Subsequent transcription and translation of this DNA sequence yields aproduct whereby the peptide mass label is fused to the protein.

[0176] F. Enzymatic Amplification Techniques

[0177] Nucleic acid amplification methods may be used to preparemass-labeled probes or to detect the presence of a target sequence. Oneof the best known amplification methods is the PCR™ which is describedin detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, eachincorporated herein by reference, and in Innus et al. (1990,incorporated herein by reference).

[0178] In PCR™, two primer sequences are typically prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. The primers may hybridize to form a nucleic acid:primercomplex if the target sequence is present in a sample. An excess ofdeoxynucleoside triphosphates are also added to a reaction mixture alongwith a DNA polymerase, e.g., Taq polymerase, that facilitatestemplate-dependent nucleic acid synthesis.

[0179] If the marker sequence:primer complex has been formed, thepolymerase will cause the primers to be extended along the markersequence by the addition of nucleotides. By raising and lowering thetemperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated. These multiple rounds of amplification, referred to as“cycles”, are conducted until a sufficient amount of amplificationproduct is produced.

[0180] A reverse transcriptase PCR™ (“rtPCR™”) amplification proceduremay be performed in order to quantify the amount of mRNA amplified.Methods of reverse transcribing RNA into cDNA are well known anddescribed in Sambrook et al., 1989.

[0181] Another method for amplification is the ligase chain reaction(“LCR”), disclosed in European Patent Application No. 320,308,incorporated herein by reference. In LCR, two complementary robe pairsare prepared, and in the presence of the target sequence, each pair willbind to opposite complementary strands of the target such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR™, bound ligated unitsdissociate from the target and then serve as “target sequences” forligation of excess probe pairs. U.S. Pat. No. 4,883,750, incorporatedherein by reference, describes a method similar to LCR for binding probepairs to a target sequence.

[0182] Qbeta Replicase, described in PCT Patent application No.PCT/US87/00880, may also be used as still another amplification methodin the present invention. In this method, a replicative sequence of RNAwhich has a region complementary to that of a target is added to asample in the presence of an RNA polymerase. The polymerase will copythe replicative sequence.

[0183] An isothermal amplification method, in which restrictionendonucleases and ligases are used to achieve the amplification oftarget molecules that contain nucleotide 5′-[alpha-thio]-triphosphatesin one strand of a restriction site may also be useful in theamplification of nucleic acids in the present invention. Such anamplification method is described by Walker et al. (1992, incorporatedherein by reference).

[0184] Strand Displacement Amplification (“SDA”) is another method ofcarrying out isothermal amplification of nucleic acids which involvesmultiple rounds of strand displacement and synthesis. A similar method,called Repair Chain Reaction (RCR), involves annealing several probesthroughout a region targeted for amplification, followed by a repairreaction in which only two of the four bases are present. The other twobases can be added as biotinylated derivatives for easy detection. Asimilar approach is used in SDA.

[0185] Target specific sequences may also be generated using a cyclicprobe reaction (“CPR”). In CPR, a probe having 3′ and 5′ sequences ofnon-specific DNA and a middle sequence of specific RNA is hybridized toDNA which is present in a sample. Upon hybridization, the reaction istreated with RNase H, and the products of the probe identified asdistinctive products which are released after digestion. The originaltemplate is annealed to another cycling probe and the reaction isrepeated.

[0186] Other nucleic acid amplification procedures includetranscription-based amplification systems (“TAS”), including nucleicacid sequence based amplification (“NASBA”) and 3SR (Kwoh et al., 1989;PCT Patent Application WO 88/10315, each incorporated herein byreference).

[0187] In NASBA, the nucleic acids may be prepared for amplification bystandard phenol/chloroform extraction, heat denaturation of a clinicalsample, treatment with lysis buffer and minispin columns for isolationof DNA and RNA or guanidinium chloride target specific sequences.Following polymerization, DNA/RNA hybrids are digested with RNase Hwhile double stranded DNA molecules are heat denatured again. In eithercase the single stranded DNA is made fully double stranded by additionof second target specific primer, followed by polymerization. Thedouble-stranded DNA molecules are then multiply transcribed by apolymerase such as T7 or SP6. In an isothermal cyclic reaction, theRNA's are reverse transcribed into double stranded DNA, and transcribedonce again with a polymerase such as T7 or SP6. The resulting products,whether truncated or complete, indicate target specific sequences.

[0188] European Patent Application No. 329,822 (incorporated herein byreference) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), single-strandedDNA (“ssDNA”), and double-stranded DNA (“dsDNA”), which may be used inaccordance with the present invention.

[0189] Following amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989).

[0190] Alternatively, chromatographic techniques may be employed toeffect separation. There are many kinds of chromatography which may beused in the present invention: adsorption, partition, ion-exchange andmolecular sieve, and many specialized techniques for using themincluding column, paper, thin-layer and gas chromatography (Freifelder,1982).

[0191] Separation may also be achieved using biologically basedinteractions such as biotin-streptavidin or antibody-antigeninteractions.

[0192] In embodiments where the mass labels have been incorporated intothe product, detection of the mass labels may be used to confirmamplification. When the mass label is to be added later, amplificationproducts should typically be visualized in order to confirmamplification of the sequences. One typical visualization methodinvolves staining of a get with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products may typically be exposed to x-ray film orvisualized under the appropriate stimulating spectra, followingseparation.

[0193] G. Chemical Synthesis Techniques

[0194] If the probe is chemically synthesized, the mass label may beplace at one or more locations within the reactive group. For example,polypeptide compounds of the present invention may be synthesized usingknown methods for peptide synthesis (Atherton & Shepard, 1989). Thepreferred method for synthesis is standard solid phase methodology, suchas that based on the 9-fluorenylmethyloxycarbonyl (“FMOC”) protectinggroup (Barlos et al., 1989), with glycine-functionalized o-chlorotritylpolystyrene resin. Solid phase peptide synthesis allows for strategicplacement of a mass label within the compound. Similarly, anoligonucleotide probe, for example, may be specifically labeled byintroducing a modified mass-labeled phosphoramidite at a particularlocation within the sequence. Chemical synthesis methods also permit theplacement of mass labels at the termini of the probe or within aninternal linker wherein the mass label is not directly attached to thebase of a nucleotide. A generalized example of a mass-labeledphosphoramidite is shown in FIG. 1B. Chemical synthesis methods for DNAare well known within the art (Agrawal, 1993)

[0195] The use of combinations of different mass labels can greatlyenlarge the number of unique mass signatures that are available whenmaking a library of nucleic acid probes, while needing only a modest setof different mass label components. As an example, usingpolymerase-based methods and a repertoire of 40 different mass-labeledthymidine triphosphate nucleotides each with a unique mass label, onemay synthesize an enormous array of differentially labeled probes. Ifcombinations of two different mass labels out of the 40 are used foreach probe then a total of 780 probes may be made each with a unique,two-mass signature [=40!/(2!.38!)=780]. If three different labels areused per probe then 9,880 different combinations are possible[=40!/(3!.37!)=9,880]. The trend continues using the example ofcombination of sets of mass labels from a pool of 40 label molecules asfollows: a set of four labels yields 91,390 possible combinations, fivelabels yields 658,008 possible combinations, six labels yields 3,838,380possible combinations and so on. Conceivably probes may be made with aunique mass label signature for every gene within humans, and any otherorganism for that matter. Examples of enzymatic probe synthesis areshown in FIG. 4C and FIG. 4D.

[0196] An alternative to the use of mixtures of mass-labelednucleotides, is the use of mixtures of mass-labeled primers. Nucleicacid probes prepared by an amplification method, such as PCR™, mayutilize mixtures of primers whereby each primer contains a differentmass label and the same DNA sequence. As with the mass-labelednucleoside triphosphates, a repertoire of mass labeled primers may beused to prepare many different mass signatures. In addition to usingmixtures of primers with a single type of mass label, primers may beprepared containing several different mass labels within a singlemolecule.

[0197] A particular advantage to the solid phase method of synthesis isthe modification of these compounds using combinatorial synthesistechniques. Combinatorial synthesis techniques are defined as thosetechniques producing large collections or libraries of compoundssimultaneously, by sequentially linking different building blocks.Libraries can be constructed using compounds free in solution, butpreferably the compound is linked to a solid support such as a bead,solid particle or even displayed on the surface of a microorganism.Several methods exist for combinatorial synthesis (Holmes et al., 1995;Burbaum et al., 1995; Martin et al., 1995; Freier et al., 1995; Pei etal., 1991; Bruce et al., 1995; Ohlmeyer et al., 1993); including splitsynthesis or parallel synthesis. Split synthesis may be used to producesmall amounts of a relatively large number of compounds, while parallelsynthesis may produce larger amounts of a relatively small number ofcompounds. In general terms, using split synthesis, compounds aresynthesized on the surface of a microparticle. At each step, theparticles are partitioned into several groups for the addition of thenext component. The different groups are then recombined and partitionedto form new groups. The process is repeated until the compound iscompleted. Each particle holds several copies of the same compoundallowing for facile separation and purification. Split synthesis canonly be conducted using a solid support.

[0198] An alternative technique known as parallel synthesis may beconducted either in solid phase or solution. Using parallel synthesis,different compounds are synthesized in separate receptacles, often usingautomation. Parallel synthesis may be conducted in microliter platewhere different reagents can be added to each well in a predefinedmanner to produce a combinatorial library. Parallel synthesis is thepreferred approach for use with enzymatic techniques. It is wellunderstood that many modifications of this technique exist and can beadapted for use with the present invention. Using combinatorial methods,a large number of unique mass-labeled probes may be synthesized.

[0199] One embodiment is an approach to synthesizing all possiblecombinations of sequence simultaneously in such a way that each uniquesequence within the pool will possess a unique mass signature. Thesynthetic approach involves the use of a unique set of four mass-labelednucleotides for each position within an oligonucleotide probe, i.e., aset of four mass labels are used exclusively at position 1, while adifferent set of four is used exclusively at position 2, and so on. Theprimary method of synthesizing said probes is chemical usingphosphoramidite chemistry though other chemical and enzymatic methodsincluding single base addition by polymerase may also be employed. As anexample, synthesis of the combinatorial set of all oligonucleotides 10bases long would require 40 different phosphoramidites, 10 different A'swith unique mass labels, 10 different C's with unique mass labels, 10different G's with unique mass-labels, and 10 different T's with uniquemass labels. The scheme is illustrated in FIG. 4A.

[0200] Utility for the complete probe set is diverse. Applicationsinclude hybridization assays for identity of cDNAs of other sequencespresent in a solid phase bound array or some other format, mappingapplications, and other diagnostic applications. It is also possible touse the set for random PCR™ amplification assays where the products areseparated by electrophoresis and the primers that paired to form thedifferent PCR™ products are identified. These applications also apply tothe methods used to identify short sequence reads.

[0201] The combinatorial synthesis of probes can be performed as asingle reaction in a single receptacle, or it may be performed using thesplit synthesis technique previously described. If the combinatorialsynthesis does not utilize split synthesis techniques, there may bedifficulties identifying sequence in cases where multiple probeshybridize. In cases where the full set of probes are used it may bedifficult to uniquely identify the sequences of the probes if more thanone probe is present at a significant level. One possible approach tolimiting the number of probes that hybridize to a particular target isby attaching a unique anchoring sequence to the probe set limiting thelocations where the probe can hybridize. This anchoring is similar tothe methods used to identify short sequence reads. As describedpreviously, it may also be possible to add extra bases to the end of theprobe to lengthen the sequence determination and improve discrimination,if necessary.

[0202] A specific example of using the anchored, combinatoriallysynthesized probes is shown in FIG. 4B. In the case of screening genomicor cDNA clone inserts, the anchored, invariant sequence may be used tohybridize to the know vector sequence immediately adjacent to the insertor in the specific case of a cDNA insert to the poly A/T region of theinsert.

[0203] For addition of labels to an already synthesized probe, hereinreferred to as post-modification, various chemically active sites on theprobe may be utilized. For example, a proper functionality of a labelcould be reacted with a primary amine on 5 propargyl amino deoxyuridine,a terminal amino or carboxyl linker, or an endogenous moiety, such asthe exocyclic amine in cytosine, guanine, or adenine. Potential linkergroups include the heteobifunctional cross-linking agent mal-sac-HNSA(Bachem Inc., Torrence, Calif.), or any of a variety of cross-linkingagents available from Pierce Chemical Company (Rockford, Ill.). Oneskilled in the art could in light of the present disclosure supply otherexamples. Post modification also allows for the addition of multiplemass labels.

[0204] I. Assays with Nonvolatile, Releasable Mass-labeled Probes

[0205] The described mass-labeled nucleic acid probes have a variety ofuses. Labeled polypeptides may be used to detect interaction of areactive group with a specific target. Representative examples include amass-labeled antibody to detect an antigen either in solution or on asolid support or a mass-labeled enzyme to detect a substrate. One ofskill in the art would recognize there are many such interactionsdetectable using labeled polypeptides to detect interactions with atarget molecule.

[0206] One preferred embodiment of the invention relates to the simpledetection of a specific target nucleic acid.

[0207] There are a variety of reasons for detecting a particular nucleicacid sequence. These reasons include, but are not limited to, detectionof infectious agents within a clinical sample, detection of anamplification product derived from genomic DNA or RNA or message RNA, ordetection of a gene (cDNA) insert within a clone. Simple detection mayemploy any combination of the methods described herein for thepreparation of the nucleic acid probe and the release and detection ofthe mass label. One may also quantify the amount detected. Most of thesemethods involve the use of a hybridization-specific event to trigger therelease of a mass label, and in cases where only small amounts of targetmaterial are present, the use of an amplification technique.

[0208] An advantage to using mass-labeled compounds that are detectableby mass spectrometry methods is the ability to simultaneously detectmany target compounds at the same time. Due to broad overlappingspectrums produced by existing fluorescent chromophores, an upper limitfor fluorescence multiplexing is most likely to be about ten differentlabels. With a matrix-assisted laser desorption/ionizationtime-of-flight (“MALDI-TOF”) mass spectrometer or directlaser-desorption mass spectrometer or an electrospray mass spectrometer,multiplexing of tens of hundreds and perhaps even thousands of differentmass labels is possible. A nonvolatile pool of labels may provide awider range of masses and structures. Due to this multiplexing ability,not only can many labeled probes be used at the same time, anyindividual probe can be labeled with many different labels.

[0209] J. Single Nucleotide Polymorphism Detection

[0210] Further embodiments involve the detection of single basevariations. These applications will generally require a great deal ofsensitivity. These applications include detection of “hot spot” pointmutations and identification of the base at single nucleotidepolymorphism (“SNP”) sites. Mass-labeled probes may be prepared thathybridize immediately adjacent to a polymorphic site and a polymerasemay then be used to add one base at the site of the polymorphism. Theparticular base may be added to the probe by many ways. For example, ina preferred embodiment where a single probe is used, a mixture of thefour chain terminating triphosphates may be added, each with a uniquemass label attached. In the homozygous SNP case only one of the fourchain-terminating nucleotides may add to the end of the probe couplingthe associated mass label to the probe. Several approaches may be takenin releasing the mass label from the probe. These approaches include,but are not limited to, the use of chemically labile functional groupslinking the mass label to the terminating nucleotide, chemically labilefunctional groups within the backbone of the extended primer or thechain-termination nucleotide, or the use of an enzyme to cleave at oneor more of the phosphodiester or glycosidic linkages within the primerextension product. In cases where the mass label release point is withinthe backbone of the extension product, the released mass label mayinclude the terminal nucleotide or some mass-modified version thereof.In another version where the release point is internal to the primerextension product, the native chain-terminating nucleotides themselvesmay serve as all or a portion of the mass labels since each basepossesses a unique mass. In cases where the mass label is chemicallycleaved from the probe, any unincorporated nucleotides may first beremoved or washed away so that they are not visualized by the massspectrometer.

[0211] Partitioning of the hybridized mass-labeled chain-terminatingtriphosphate may be done on the basis of mass differences, as labeledtriphosphate hybridized to a target-hybridized probe will have a highermolecular weight than a labeled triphosphate that is not. The probe ortarget may also be attached to a solid-phase via a number of meansincluding biotin/streptavidin or chemical coupling or UV cross-linking.An alternative is the use of a nuclease to digest the mass-labeledprobe. Using a nuclease the mass labeled chain-terminating nucleotidewill be released as a monophosphate. The unincorporated mass-labeledchain-terminating nucleotides will remain as triphosphates, and theresulting mass shift to monophosphate will indicate which nucleotide wasincorporated. This nuclease method relieves the necessity to removeunincorporated nucleotides prior to analysis.

[0212] Another embodiment encompasses the multiplexing of a large numberof probes so as to detect many SNPs simultaneously. Preferably masslabels may be present to uniquely tag each of the probes that comprisethe pool. The addition of a biotinylated chain-terminating nucleotide atthe site of the point polymorphism may also be used to segregate theprobe population depending on which probes incorporate a specificbiotinylated chain-terminating nucleotide and which do not. As anexample, the pool of mass-labeled probes with target may be divided intofour reactions. The first reaction would contain only biotinylateddideoxy adenosine triphosphate, the second would contain onlybiotinylated dideoxy cytidine triphosphate, the third only biotinylateddideoxy guanidine triphosphate, and the fourth only biotinylated dideoxythymidine triphosphate. Following a single base extensionpolymerase-dependent reaction in the presence of the proper nucleotide,the extended products are captured, washed and the mass labels arereleased for mass spectrometric analysis. In the first reaction onlythose mass-labeled probes that incorporate an A will be visualized. Inthe second reaction only those mass-labeled probes that incorporated a Cwill be visualized. For the third and fourth reactions probes thatincorporated, respectively a G or a T will be visualized. It is expectedthat hundreds of probes could be multiplexed in this way.

[0213] A person skilled in the art could identify a number of variationsof the single or multiplexed probe approach for reading out the SNPbased on either the absence or appearance of the mass label or masschange occurring in the mass label. Another example of mass changewithin a mass label is the case where the mass label is present at the3′ end of the probe. Following polymerase-dependent base extension, themass label may be released, including the chain terminating baseaddition as well as the penultimate base. A possible structure for thistype of probe is shown in FIG. 2. Placement of the mass label and therelease site may be at other bases with a preference of placement nearthe 3′ end. In all cases the mass label should preferably be placedbetween the release group and the 3′ end. In other embodiments it may bepreferred to perform what is effectively a short chain terminatedsequencing reaction, where, in addition to dideoxy nucleotides, someamount of normal deoxy nucleotides are present. Extension of the primerwill result in a nested set of products, each being chain terminated bya dideoxynucleotide correlating to its complementary base on thetemplate strand. In the preferred form, the mass label may be locatedwithin the primer near the 3′ end which contains a chemical releasegroup. Such a method offers a separate embodiment for short sequencereads as well as detection of one or more SNPs. All of the SNP detectionmethods described above may involve the use of mass modified forms ofthe different nucleotides in order to enhance the mass differencebetween the different possible products.

[0214] An alternative preferred embodiment to single base addition fordetecting an SNP is the performance of a discriminating exonucleaseevent in the presence of matching and mismatching oligonucleotideprobes. One example of this approach is to combine the use of releasablemass labels with nick translation PCR™. In addition to its polymeraseactivity, Taq DNA polymerase has both 5′ to 3′ exonuclease andendonuclease activities. If a fully complementary oligonucleotide probeis placed in the path of polymerization, for example during PCR™amplification, the polymerase will attack the 5′ end of the probe withits exonuclease activity, digesting the molecule until it is too smallto remain hybridized. However, if the oligonucleotide is not perfectlycomplementary near the 5′ end, e.g., a mismatch is present nearby, thenthe end of the probe will fray and be attacked by the endonucleolyticactivity of the polymerase rather than the exonuclease activity. Thenucleolytically cleaved product, preferably containing the mass label,will have a different final mass depending on whether or not a mismatchwas present and how the nuclease cut in response to this mismatch. Ithas been demonstrated that the initiation of endonucleolytic activitycan be influenced by the presence and placement of a mismatch within thehybridization probe (Holland et al., 1991; Lee et al, 1993). Selectiveplacement of a mass label within the oligonucleotide probe relative tothe expected mismatch site can be used to yield a differential signaldepending on whether or not an actual mismatch is present.

[0215] By taking advantage of the high multiplexing capability ofmass-labeled probes, one can extend this assay to the simultaneousdetection of multiple SNPs. Each of the probes targeting a particularSNP contains one of the four possible bases to complement the site ofpolymorphism. The placement of the mass label is such that if the probecontains a perfect match tot he template, the mass label will bereleased by the exonuclease activity of Taq polymerase, primarily in aform that includes a single nucleotide. The other probes will create amismatch and the endonuclease activity of the polymerase will initiatecutting of the probe in such a way that the mass label remains bound toa larger segment of the probe that includes more than one nucleotide.The shift in mass of the mass label cleavage product is diagnostic ofwhether or not a mismatch has occurred.

[0216] When the detection by mass spectrometry is performed using MALDIit may be possible to select a matrix that can visibly discriminatebetween the smaller product that results from the matching probe and thelarger product that results from the mismatched probes such that thesmaller product is desorbed more efficiently or selectively. Utilizing amatrix such as 2,5-dihydroxybenzoic acid, sinapinic acid, orα-cyano-4-hydroxycinammic acid, the signal strength decreases as morenucleotides are attached to the probe (Jensen, et al., 1996).

[0217] By using a set of 50 mass-labeled probes, as many as 25 biallelicSNPs may be detected in a single tube. As is the case with any PCR™based detection scheme, the limit of SNPs to be detected will morelikely be the result of the limits of multiplexing PCR™. The process,when coupled to high throughput mass spectrometric analysis, can beespecially cost efficient when analyzing a small set of polymorphicsites, e.g., in a cluster of exons, as part of a population study wherethousands to tens of thousands of samples need to be analyzed.

[0218] Nick translation PCR™ combined with mass-labeled probes can alsobe used as a generalized method for the detection and monitoring of aPCR™ amplification reaction. In this case, only matching probes arepresent and the mass label is released only if PCR™ of the particularregion targeted by a particular probe is amplified.

[0219] While the preferred embodiment for these assays is to usenonvolatile releasable mass labels or involatile releasable mass labels,other types of labels can be used as well, such as isotopic mass labels,volatile mass labels (including electrophores), fluorescent labels, andchemiluminescent labels.

[0220] K. Short Sequence Reads

[0221] In another preferred embodiment of the invention, themass-labeled probes may be used to identify short sequences. Inparticular, combinations of hybridization and enzymatic (polymerase orligase) extension can be employed with the labeled probes to identifyshort sequence runs adjacent to a “priming” or anchoring region. Thereare three optimal methods for doing this. The first method isillustrated in FIG. 3A. A mixture of probes are synthesized containingtwo domains, a fixed sequence recognition domain, typically comprised ofonly one or a few sequences, and a randomized domain, comprising thefull set (or some subset) of all possible sequences. The fixed sequenceof the probe is used to target hybridization of the probe to a singlesite within a particular target nucleic acid. This target site istypically invariant. The sequence adjacent to the invariant sequence isvariable and, depending on the particular target, can have any one ofthe total combinations of sequence. In order to probe for allpossibilities it is necessary to synthesize probes containing all thepossible secondary domain sequence combinations. If the second proberegion is four bases in length, then 256 different probes need to besynthesized. If the second probe region is five bases in length, then1024 different probes need to be synthesized. Six bases required 4096,and so on. The probes can be synthesized individually, each possessing aunique combination of mass labels as a releasable mass signature.Alternatively, the probes can be synthesized with unique mass signaturesusing a combinatorial synthesis method of the type described previously.In particular embodiments regarding diagnostic probes, it may bedesirable to generate only a small number of probes, for example lessthan 20.

[0222] The two domain probes are useful for identifying the end sequencewithin clone inserts. As an example, the fixed sequence domain wouldhybridize to the cloning vector sequence immediately adjacent to theinsert sequence. The variable sequence is then available to hybridize tothe cloned insert. Only the probe that is complimentary to the clonedinsert sequence adjacent to the cloning vector sequence will form aperfect hybrid. The remaining two domain probes will not. Detection ofthe mass label signature for the probe that has hybridized using one ofthe methods described will identify the probe sequence and the cloneinsert sequence. Other applications include targeting hypervariablesequence regions or mutation/polymorphism analysis at targeted sites. Inall cases the fixed sequence of the probe directs the probe to a uniqueregion within the target, essentially anchoring where the variableregion will probe.

[0223] In order to increase the level of discrimination and extend theread length for the short sequence read it is possible to use an enzyme,such as polymerase or ligase, to add a single nucleotide oroligonucleotide to the end of the variable region of the anchored probe,optionally including mass labels on the added nucleotide oroligonucleotide that can identify the sequence for these additions.Addition of bases by either enzyme places stricter requirements on thevariable region being a perfect hybrid to enable enzymatic action.Examples of how these probe additions work are shown in FIG. 3B. Notethat for polymerase the addition needs to be to the 3′ end of the probewhile ligation can occur at either the 3′ end of 5′ end. As with thevariable region within the probe increasing size of the addition willnecessitate a larger and larger pool to represent all possiblesequences. Oligonucleotide additions don't necessarily need to beentirely variable. There may be cases where the variable region willcontain an invariant region. Such extensions will increase thethermodynamic stability of the oligonucleotide addition and allowligation to occur at higher temperatures. It is also possible toenvision cases where invariant nucleotide sequence would be intermingledwith the variable sequences described.

[0224] Combinatorial libraries may also be used to detect shortsequences. In cases where the full set of probes are used, though, itmay not be possible to uniquely identify the sequences of the probes ifmore than one probe is present after hybridization at a significantlevel. One possible approach to limiting the number of probes thathybridize to a particular target is attaching a unique anchoringsequence to the probe set limiting the locations where the probe canhybridize. This anchoring is similar to that previously described foranalysis of short sequence reads. As previously described, it is alsopossible that extra bases could be added to the end of the probe tolengthen the sequence determination and improve discrimination, ifnecessary.

[0225] A specific example of using the anchored, combinatoriallysynthesized probes is shown in FIG. 4B. In the case of screening genomicor cDNA clone inserts the anchored, invariant sequence is used tohybridize to the known vector sequence immediately adjacent to theinsert or in the specific case of a cDNA insert to the poly A/T regionof the insert.

[0226] While the preferred embodiment for these assays is to usenonvolatile releasable mass labels or involatile releasable mass labels,other types of labels can be used as well, such as isotopic mass labels,volatile mass labels (including electrophores), fluorescent labels, andchemiluminescent labels.

[0227] L. Targeted Cleavage Mismatch Detection

[0228] It is of interest to detect the presence of a mutation within agiven sequence in cases where one does not have prior knowledge ofexactly where the particular mutation might occur. Oligonucleotideprobes may be used for hybridization to a target DNA containing a singlemutation within a region of interest, leading to the formation of amismatch. In one embodiment of the invention, enzymatically synthesizedmass-labeled probes blocked from double-strand-specific enzymaticdigestion at the 3′ end are used. The 3′ ends of the probes can beblocked by chemical modification or enzymatically. For example, blockingcan be achieved by making the 3′ terminus inaccessible to enzymaticdigestion. After hybridization of the probe to the target sequence,treatment with a mismatch specific chemical or enzymatic cleavingreagent would cleave the hybridized pair at the mismatch site.Representative cleaving regents include KMNO₄ and T4 endonuclease VII.Subsequent treatment of the cleaved pair with a double-strand-specific3′-5′ exonuclease, such as exonuclease III, would lead to digestion ofprobe from the cleavage site to the 5′ labeled end, thereby releasingthe mass label. This method is illustrated in FIG. 5A and FIG. 5B. As analternative, the polarity of the system can be reversed by placement ofthe mass label at the 3′ end of the probe and by using adouble-strand-specific 5′-3′ exonuclease, such as T7 gene 6 exonuclease.

[0229] Another example of mismatch detection involves the amplificationof heterozygous target DNA using two different mass-labeled probes. Thedifference can be a single base mutation, for example A:T to G:C. Fourproducts are produced by the PCR™ reaction, two fully homogenousproducts representing the original sequences, while the other twoproducts contain a mismatch at the mutation site. Treatment withterminal transferase adds long 3′ overhangs to all of the products.Chemical or enzymatic mismatch specific cleavage is used, affecting onlythe two heterogeneous pairs. Exonuclease III digestion also affects onlythe cleaved heterogeneous pairs, releasing the mass labels withoutdigesting the sequences blocked by the 3′ overhangs. This method isshown in FIG. 5C and FIG. 5D. These mismatch methods could also becombined with other labeling methods such as fluorescent tags orradiolabels.

[0230] While the preferred embodiment for these assays is to usenonvolatile releasable mass labels or involatile releasable mass labels,other types of labels can be used as well, such as isotopic mass labels,volatile mass labels (including electrophores), fluorescent labels, andchemiluminescent labels.

[0231] M. Highly Multiplexed Probe Screening Assays

[0232] A number of novel applications become possible with multiplexed,mass-labeled probes where the preferred mode is to be able to screen alarge number of targets simultaneously. Multiplexed applications includemultiple pathogen diagnostics, multigene genetic polymorphism screening,SNP genotyping, clone and gene mapping, and gene expression analysis.

[0233] Highly multiplexed analysis by hybridization can be categorizedinto one of three approaches: (A) hybridization of a library of probeswith known sequence against a library of targets of unknown sequence,(B) hybridization of a library of probes with unknown sequence against alibrary of targets of know sequence, and (C) hybridization of a libraryof probes with unknown sequence against a library of targets of unknownsequence.

[0234] Approach (A) is beneficial for applications such as diagnostics,genotyping, expression analysis and probe mapping where it has beenpredetermined what sequences are to be screened. Many of the methodsdescribed above may be used in approach (A). Combinatorially synthesizedprobes can be used with approach (A) where the sequences of the probes(and target to which the probe is hybridized) are postdetermined, i.e.probe and then determine the sequence of which probe has hybridized. Thelimits as previously described for combinatorial probes apply. Use ofrepertoire sets of mass labeled probes, as opposed to combinatorialprobes, can be used in multiplexed mixtures to detect the presence ofshort sequences for purposes of sequencing by hybridization or producinga probe signature for a particular target sequence.

[0235] Approach (B) provides a path for a number of applications where alibrary of different known DNA sequences, such as oligonucleotides, PCR™products, RNA transcripts or DNA clones, have been arranged and areavailable for partitioning the unknown probe set. These methods often,but not always, include the use of solid phase arrays to physicallypartition the known sequences prior to probing. Applications includecompetitive hybridization for differential expression analysis and fastmapping of genes, subclones or short sequence tags (SSTs) against amaster genomic clone library, multiplexed infectious agent detection orany other set of samples that need to be probed in a multiplexedfashion.

[0236] Approach (C) is useful in cases where it is not necessary to knowsequence but only to determine trends. As an example, one might want todetermine the degree of homology or complementarity between two or morespecies or two or more expressed gene sets. Random or semirandom probesagainst random or semi random target can provide percentage values forhomology. In these cases probes or targets that exhibit differentproperties, e.g., fall into the nonhomologous category, may be taken onfor further analysis to determine their sequences. Such a method couldbe used for gene discovery.

[0237] A practical example employing these approaches is in measuringgene expression profiles. The most basic way to measure a geneexpression profile is statistically, to count the number of message RNAs(mRNAs) produced for each particular gene within a particular cellularsample. The more mRNA copies of a particular gene, the higher its levelof expression. The approach commonly taken is to separate out arepresentative number of mRNAs through a process of copying the mRNA tocomplementary DNA (cDNA), and then growing up the individual clonecolonies of each cDNA on culture plates. Typically, cDNAs are cloned byinsertion into either a plasmid or a phagemid cloning vector, and thentransformed into bacterial or encapsidated into phage respectively. Eachclone represents an individual mRNA derived from the total population.The set of clones comprises a gene expression library.

[0238] Currently, the common approach used in genomic research to screenthe clones and to identify which mRNA/gene correlates to which clone isto sequence the DNA. A portion of each cDNA clone sequence is readcreating an expressed sequence tag (EST) that uniquely identifies themessage/gene sequence. Identity is made by comparing the EST to genomicdata bases containing previously identified gene sequences. In severalyears, all human EST sequences will be placed into existing public andprivate databases.

[0239] When screening a particular clone library, possibly a librarythat includes 10.000 clones, any particular EST may appear multipletimes. The more times a particular EST appears, the higher theexpression level for the gene correlating to the EST. The more clonesthat can be read, the more statistically representative the EST datawill be to actual expression. Screening larger numbers of clones alsomakes it more likely that genes expressed at low levels will beidentified.

[0240] With this in mind, it would be ideal to be able to screen 100,000or more clones per library. However, this level is costly andimpractical using existing sequencing technology. Typical sequencingscreens analyze 500-10,000 samples at a cost of $5,000 to $100,000. NewDNA sequencing technology will be able to lower this cost somewhat.

[0241] The mass-labeled hybridization probes of the present inventioncould simplify and lower the cost of gene expression analysis. The probeapproach primarily utilizes knowledge of the genes,to be analyzed. Sincethe vast majority of gene sequences will be known within a few years, itis not necessary to use a de novo technique. It is also possible todetect previously unknown genes with these hybridization procedures.Complete identification of new genes may require a separate DNAsequencing analysis, subsequent to a hybridization assay, to determinethe sequence of any of these newly discovered genes.

[0242] As is the case for the sequencing-based approach to geneexpression analysis, the hybridization approaches of the currentinvention will usually involve converting the mRNA population to cDNA,transforming the cDNA into bacteria and growing bacterial colonies onculture plates and screening bacterially derived plasmids. Following theprocess of approach (A), hybridization of a library of known probesagainst a library of unknown targets (the cDNA clones), the clones to bescreened can be spotted in a regularly spaced array or grid on a surfacesuch as a nylon filter, glass, silicon or gold. The typical processinvolving bacteria colonies involves lysing the bacteria cells on thegrid and fixing the DNA to the surface. The grid of cDNAs represent thelibrary of tens to hundreds of thousands of expressed messages to beprobed.

[0243] In conventional methods, a grid can be probed with only onesingle probe sequence at a time, typically being radioactively labeledas shown in FIG. 9A. Following the gridding of the unknown cDNAs, thelibrary cDNA array is wetted with a solution containing the labelednucleic acid probe. The grid-probe solution is incubated to allow theprobe to hybridize its complement at one or more positions within thegrid. Following hybridization, the grid is imaged in order to locate theprobe-hybridization positions. In order to use multiple probesrepresenting multiple genes, the grid needs to be replicated and adifferent grid is used for each probe. Using fluorescent labels, fourdifferent chromophores can be multiplexed within a sample andindividually detected with the aid of software deconvolution of thefluorescence emission spectrum as shown in FIG. 9B. However, thepractical upper limit for fluorescence multiplexing is likely to bearound 10 different labels due to the broad overlapping spectrumproduced by existing fluorescent chromophores.

[0244] Use of releasable, nonvolatile mass labels to uniquely labelindividual probes provides a means of using a highly multiplexed set ofprobes to simultaneously screen a single grid of unknowns. The nucleicacid probes can be synthesized using individual cDNAs with knownsequence as templates. In all cases the probes may use combinations ofmass labels or single mass labels. Following synthesis andmass-labeling, the different probes can be combined and used to probe asingle grid in a multiplex fashion. The probing procedure is identicalto that used for a single radioactively labeled probe until the imagingstep is reached. Instead of using a phosphorimager or x-ray film, thegrid is scanned within the mass spectrometer after release of thelabels, pausing briefly at each position to detect the mass label signalthat may be present.

[0245] The number of probes used is only limited to the number of probesone is willing to make and to the number one is interested in. As anexample, one may be interested in a set of 1000 genes that may play animportant role in a particular disease or one may wish to look at 50.000different genes. In either case the probes may be individuallysynthesized or produced in combinations in microliter plates usingliquid handling robotics. Likely approaches include the performance ofT7 RNA polymerase transcriptions of plasmids containing known cDNAinserts using mass-labeled nucleoside triphosphates to producemass-labeled RNA probes, PCR™ reactions amplifying known cDNA insertsusing either mass-labeled nucleoside triphosphates or mass-labeled DNAprimers to produce mass-labeled DNA probes or chemically synthesizedmass-labeled oligonucleotide probes. Examples of enzymatic probesynthesis are provided in FIG. 4C and FIG. 4D. Within each synthesisreaction a different single or unique combination of mass-labelednucleoside triphosphates are added which thereby incorporate a uniquemass signature within each newly synthesized probe. In the cases ofmass-labeled oligonucleotide probes it is also possible to usechemically synthesized combinatorial probes. Following synthesis, theprobe set is mixed together to create a master probe mix. A number ofmater probe mixes can be prepared to perform multiplexing if desired,where each cDNA of each master probe mix has a unique combination masslabel signature. The probe set or sets can then be used to probe a largenumber of different unknown complementary DNA gridded libraries as shownin FIG. 9C. Different libraries can be prepared from a variety ofsamples, for example exposed to different stressor conditions and/ordifferent est pharmaceuticals, possibly with time as an additionalvariable.

[0246] An alternative method for gene expression analysis follows theprocess of approach (B), hybridization of a library of unknown probesagainst a library of known targets sequences. Rather than uniquelylabeling known gene probes to hybridize against unknown cDNAs, one canlabel libraries of unknown cDNAs and hybridize against known unlabeledgene probes arrayed on a grid. This method has been described for twolibraries using fluorescently labeled unknown cDNA mixtures (Schena etal., 1995; incorporated herein by reference) as shown in FIG. 10A. Inthe fluorescent case, first strand cDNA is prepared form two separatecellular samples. Synthesis of the first mixture of cDNAs is performedin presence of one particular fluorescent nucleotide, and the synthesisof the second mixture in the presence of a different fluorescentnucleotide. The mixture of cDNAs, which reflect the relative abundanceof different mRNAs from each sample, are then mixed and allowed tocompetitively hybridize to a gridded array of known genes present on asolid phase surface. After the cDNAs have hybridized to the grid, andunbound labeled cDNAs are washed away, the relative fluorescenceintensity for the two dyes is measured at each position in the griddedarray. If the fluorescence intensity for each dye is equivalent then thecorresponding mRNAs from each sample were expressed at a similar level.If the fluorescence intensity is stronger for one dye than the other ata particular position/gene in the gridded array, then that gene wasexpressed at a higher level in the sample whose fluorescence wasstronger.

[0247] By utilizing the mass labeling methods to prepare the cDNAs,rather than fluorescence, it is possible to prepare and simultaneouslyhybridize cDNAs from many different cellular sources to the griddedarray of known genes. Instead of only two or three cDNA pools beingcompared simultaneously, the use of mass labels makes it possible tocompare tens if not hundreds of cDNA pools simultaneously as shown inFIG. 10B. The mass labels can be released by any of the appropriaterelease mechanisms described and the grid can be scanned for the masslabel signal. The intensity of the mass signals at a given grid positionwill be proportional to the level of mRNA in the original sample thatcorresponds to the detected cDNA on the grid. The relative ratios of thecompeting mass labels are determined providing information about thedifferences in gene expression between all of the different samples forall of the genes present on the gridded array.

[0248] This same multiplexed mass-labeled probe methodology can be usedto quickly map genes to large genomic libraries. Gridded libraries ofP1, PAC/BAC and YAC clones can be prepared in the same manner as cDNAfilters. Multiple label studies provide a means for quickly mappinggenes and identifying gene clusters. Probes generated from particularclone inserts or gene sequences are used to screen libraries of genomicor cDNA clones. Hybridization events indicate an overlap of insertsequence in the genomic case and the presence of a gene in the cDNAcase. These libraries can also be used for intergenomic probing, e.g.,probing a C. elegans library with human gene probes, and visa versa.

[0249] The technology for probing with and detecting mass labels withingridded arrays can also be applied to other solid phase systems whereDNA probes are utilized, specifically Northern and Southern assays. Inthese two methods the initial phase is to run a polyacrylamide gel andthen to transfer the DNA to a nylon membrane using a blotting procedure(Sambrook et al., 1989). As with other procedures described above,mass-labeled nucleic acid probes can be prepared to hybridize to thefilters. In another embodiment mixtures of single or combinations ofmass labels can be used in an effort to multiplex the detection. A scanof the filter after hybridization and washing within the massspectrometer provides the means to detect, and where necessary quantify,the amount of mass label present in a particular location.

[0250] An additional embodiment of the technology is the use of masslabeled protein probes, in the form of antibodies, for hybridizationagainst one and two-dimensional protein gels. One skilled in the art canalso envision other combinations of mass labeled probe moleculeshybridized against targets bound to a solid phase matrix. In all casesthe mass label is released and either the solid phase surface analyzedusing a scanning mass spectrometer, or a transfer to another surfacetakes place before mass analysis.

[0251] Attachment of the genetic target or other target to a filter orother form of grid is not necessary as part of the broadest embodimentsof the invention. For example, a mass-labeled probe set may be directlyhybridized to DNA or RNA targets in solution. In order to discriminatebetween the probes that hybridize and the probes that do not, one of twopossible events needs to occur. Either the mass labels on hybridizedprobes need to be enzymatically released using a double-strand-specificnuclease, such as exonuclease III, lambda exonuclease, T7 gene 6exonuclease or a restriction endonuclease, or some partitioning eventneeds to occur wherein unhybridized probes are separated from hybridizedprobes. One of skill in the art can envision several means forpartitioning other than pre-binding of the target to a solid phase arrayas described in the methods above, such as hybridized probe extension bya polymerase using biotinylated nucleotides, or coupling the masslabeled probe to a biotinylated probe as part of an amplification event,such as PCR™ or LCR.

[0252] For both the nuclease case and the partitioning case, anamplification event can be used to produce a significant amount of masslabel. Mass labels attached to a probe hybridizing downstream from oneof the PCR™ primers can be released during PCR™ amplification using thenick translation 5′-3′ exonuclease activity of the thermostablepolymerase. Mass labels within primers can be released using a 5′3′exonuclease such as T7 gene 6 exonuclease after amplification. Inembodiments where a mass labeled primer is coupled to a biotinylatedprimer during amplification, or biotin is incorporated through the useof biotinylated nucleotides, and the product is partitioned away fromthe unincorporated primers, it is possible to use nonspecific cleavage,such as chemical cleavage methods, to the release of the mass label.

[0253] In another embodiments, hybridization-specific nuclease digestioncan also be used to cleave a probe containing both biotin and masslabel, in an assay where solid-phase-bound steptavidin is used to removeuncleaved mass labels. Examples of such cleavage involve the use of adouble-strand-specific nuclease such as those described above.Restriction endonucleases may be used to cleave a probe that contains arestriction site in the center and a mass label and biotin at opposingends of the probe. Another example, where RNA is used as a probe,involves double-strand-specific cleavage using RNase H.

[0254] In another exemplary method for the detection of an amplifiedsingle-stranded target such as that produced by T7 RNA polymerasetranscription, a double-stranded probe is prepared with the mass labelbeing attached to the strand that is homologous in sequence to thetarget strand. The mass-labeled strand is then displaced by acompetitive hybridization with target and the mass label is released bya single-strand specific exonuclease such as exonuclease VII, Mung Beannuclease or nuclease S1. An alternate method would employ the use ofsingle-strand specific chemical cleavage reagent to release the masslabel from a chemically modified probe. Examples of chemicalmodifications that would provide single-strand specific release of masslabel include cleavage of a ribonucleotide base by transesterification,a phosphoramidate cleavable by acid, and a 5′-P-S phosphorothioatecleavable by silver nitrate as described in Example 9.

[0255] PCR™ can also be combined with the use of a mass labeled primerand a restriction enzyme to enable release of a mass label only ifamplification occurs. In this embodiment the mass labeled PCR™ primercontains the sequence for a restriction site that becomesdouble-stranded only as part of the amplification process. Once the siteis double stranded, it is recognized by the restriction enzyme andcleaved. The cleavage event releases the mass label from bulk of theprimer and PCR™ product allowing it to be uniquely detected.

[0256] An embodiment of the invention where mass-labeled probes can beused to measure mRNA levels in solution is shown schematically in FIG.14. A series of gene-specific, mass-labeled probes (1-100 per study) areadded to the mRNA pool (or more likely, first-strand cDNAs derived fromthe mRNA pool) and allowed to hybridize. Each gene-specific probecarries a unique mass label, and possibly multiple copies of that labelto increase sensitivity. The hybridized mixture is treated with adouble-strand-specific exonuclease that releases the mass labels for theportion of the probe population that was hybridized to target genes.Only if the mRNA from a gene of interest is present will thecorresponding mass label be released and detected. In addition, thesignal intensity for the particular mass label will be proportional tothe relative abundance of the particular mRNA within the pool.Comparisons of the relative intensities for the different mass labelsreflect the relative mRNA expression levels. The relative geneexpression pattern for as many as 38,400 genes could be probed for in asingle 384 microliter plate if 100 different probes per will are used.Conversely, a set of 100 genes be examined for 384 different samples ina single microliter plate experiment.

[0257] There are examples where the mass spectrometric sensitivitylevels may be found to be insufficient to directly monitor the mRNAlevels, e.g., due to small numbers of cells as a result of poor cellgrowth, or in animal model samples derived from very small tissuebiopsies. For such samples, it may be necessary to incorporate messageamplification schemes into the methodology.

[0258] As described earlier, the use of nucleases that digestmass-labeled nucleic acid probes when they are hybridized to a targetnucleic acid affords the possibility for linear amplification of signal.In cases where the target DNA is single stranded and significantlylonger than the probe being used, it is possible to selectively digestonly the probe. Digestion of the oligonucleotide probe makes the targetstrand repeatedly available for multiple rounds of hybridization anddigestion. This type of amplification can readily achieve 2 to 3 ordersmagnitude of amplification.

[0259] Because any given study may only monitor a relatively smallnumber of genes, e.g., 20 to 100, it may be possible to use one or a fewmultiplexed PCR™ reactions to amplify only the targets associated withthe probe set. The use of PCR™ or other amplification methods mayrequire the development of additional controls so as to reduce theinfluence of amplification artifacts. The multiplexing ability ofmass-labeled probes makes it easy to include one or more controls. Theuse of redundant or semi-redundant primers, such as those used indifferential display techniques, may also provide an effectiveamplification route. In all cases where a polymerase is used foramplification, such as Taq DNA polymerase, the 5′ to 3′ exonucleaseactivity can be used to digest the probe while amplification continues(Holland et al., 1991).

[0260] All of the solution phase methods, including methods that utilizepartitioning, described above may be utilized as a means for couplingthe release of a mass label to the presence of a particular mRNAsequence. Other methods that may be used in amplification, of themessage population include ligase chain reaction, in vitro transcriptionof the cDNA population, and variants of methods for producing cDNAlibraries, such as single-well polyclonal cDNA plasmid growth.

[0261] As the full gene set of an organism becomes available, it isconceivable to prepare beforehand the complete set of mass-labeledprobes for gene expression analysis. With probes being enzymaticallysynthesized, a large stock of these probes can be made at a relativelyinexpensive cost in less than a week of effort. It is also possible toquickly make a repertoire of mass-labeled probes through chemical means.

[0262] While the preferred embodiment for the assays described herein isto use nonvolatile releasable mass labels or involatile releasable masslabels, other types of labels can be used as well, such as isotopic masslabels, volatile mass labels (including electrophores), fluorescentlabels, and chemiluminescent labels.

[0263] N. Multiplexed Mass Label Substrates in Affinity Assays.

[0264] The methods disclosed herein may also be employed in indirectschemes for identifying the presence of one or more target biomolecules.Indirect schemes, such as enzyme-linked immunosorbent assays (ELISAs),provide a method for utilizing substrate conversion to a productmolecule via enzymatic turnover of the substrate. Enzymatic catalysis ofa substrate leads to the linear amplification of the product's signal.

[0265] In an ELISA the target molecules, generally bound to the solidphase, are recognized by an antibody which noncovalently binds to thetarget. The recognition antibody is conjugated to an enzyme used tocatalyze substrate conversion to product. Traditional ELISA techniquesutilize small organic molecule substrates that when converted to productby an enzyme, such as alkaline phosphatase, horse-radish peroxidase, orurease, yield a molecule with changed optical qualities, e.g., thesolution becomes colored or the product possesses strong fluorescence.In addition, the conversion of substrate to product often produces achange in mass, thus the product may act as a mass label that may bedetected by mass spectrometry. The amount of product may be quantifiedeither absolutely or relative to the substrate used, with knowledge ofenzyme turnover rates and reaction conditions, and used to calculate theamount of a target molecule present in the assay.

[0266] Methods for traditional ELISA assays are well established (seeCurrent Protocols in Molecular Biology Vol. 2, Chapter 11, incorporatedas a reference herein). Multiple protocols exist, which includeindirect, direct competitive, antibody-sandwich, doubleantibody-sandwich, direct cellular, and indirect cellular assays. Themass label modification envisioned in this application would be designedto measure unknown quanties of target biomolecules by adaptation of thetraditional ELISA methods. In this modification, target biomolecules arecovalently or noncovalently bound to a surface, such as on a bead or aplastic dish, either directly or through a small “capturing” molecule(ligand) or a protein (such as an antibody). The target biomoleculecould also be a component of a cell that could be bound to the surfaceof the vessel. The solid-phase target biomolecules are incubated with atarget recognition molecule (antibody, ligand, oligonucleotide, etc.)that has a specific affinity for the target biomolecule. This targetrecognition molecule is conjugated to an enzyme. Four multiplexed assayseach target recognition molecule much be covalently linked to an enzymewith a unique catalytic activity for differentiation of the differenttargets (typical of the “direct” assay protocols). These conjugatedtarget recognition molecules are allowed to bind to the substrate;unbound molecules are removed by washing, then the enzyme substrates areadded under conditions in which bound enzyme reacts with its substrateto release a product with a unique mass that is detectable using massspectrometry.

[0267] “Capture antibodies” with high specific binding affinity for theantigens may be needed for soluble antigens. Methods for preparation ofspecific antibodies for either capture or quantitation of antigens arewell established in the literature. Methods for conjugating enzymes toantibodies are also well established and may include crosslinking agentssuch as glutaraldehyde or conjugation via perioxidate oxidation.Purified DNA restriction enzymes are commercially available. New enzymeswith unique catalytic activity may also be engineered using establishedmolecular procedures.

[0268] The ease of detection of a multiplex of mass labels offers theopportunity for the performance of a multiplex of immuno assayssimultaneously within a single solution. Different enzymes, conjugatedto antibodies or other target recognition molecules, used in combinationwith a set of enzyme-specific substrates may be used to yield enzymaticproducts that are unique in mass and therefore uniquely detectable andquantitatable by mass spectrometry.

[0269] In addition to multiplexing an unrelated set of enzymes andsubstrates, classes of enzymes that modify a class of substrates mayalso be multiplexed. For example, classes of enzymes all recognizing thesame substrate but modifying it in different ways may be employed as mayenzymes which recognize and modify particular chemically-relatedsubstrates, where the variations in structure alter the specificity ofparticular enzymes for the particular substrate.

[0270] A class of enzymes all recognizing the same or a few substratesis proteases. Proteases recognize different amino acids or amino acidsequence motifs and cleave the amide linkage yielding two or morefragments. Examples of proteases and their specificities include:trypsin, which cleaves at the C-terminal side of both arginine andlysine residues; thrombin, which cleaves at arginine; Glu-C, whichcleaves at the C-terminal side of glutamic acid residues; Lys-C, whichcleaves at the C-terminal side of lysines; and Asp-N, which cleaves atthe N-terminal side of aspartic acid residues. Small polypeptidescontaining specific amino acids and/or amino acid sequence motifs may beused as substrates for proteolytic digestion. The use of one or a fewpolypeptides that are recognized and cleaved differently by differentproteases sets a situation where there is a competition for substrate.The use of competitive substrates, and measurements of the relativeratios of different products derived from the same substrate, mayprovide a more accurate measure of the relative quantities of differenttarget biomolecules.

[0271] One potential problem with the use of proteases is their possibledigestion of antibodies and other proteins required for the bioassay.This problem may be overcome through a variety of means including,careful selection of proteases, selective chemical modification to blockproteolysis, and use of protease inhibitors including those that can becompetitively displaced by the reaction substrates. Alternatively,proteases may be used on other nonprotein-based assays such as probingfor nucleic acid using oligonucleotide probes conjugated to theproteases. Other classes of enzymes that may be used instead ofproteases include kinases which phosphorylate their substrates andnucleases.

[0272] Ribonucleases and deoxyribonucleases have varying specificity.Endonucleases such as RNas T1, Rnase U2, and Rnase CL3, target G, A, andC nucleotides, respectively. In a similar manner to the use of smallpolypeptides as substrate for proteases, small oligonucleotides may beused with nucleases. Nuclease resistant nucleotides, such asphosphorothioates, methylphosphonates, boranophosphates, and peptidenucleic acids can be incorporated into the substrates to direct thespecificity of the different nucleases toward yielding unique products.Unlike peptides which can be simply and easily detected by massspectrometry it may be preferred to modify the oligonucleotides with theaddition of polypeptides or other molecules to improve and ease analysisin the mass spectrometer.

[0273] Another class of enzymes is restriction endonucleases. Use ofrestriction enzymes falls under the second case described above, wheresubstrates may be chemically related but variations in structure altertheir specificity as far as to which enzyme in the class will recognizeand modify it. In this case the structural alterations are changes inthe sequence of the substrates. The substrates themselves are smalldouble-stranded oligonucleotides which contain one or more restrictionendonuclease recognition and cleavage sites. Similar to the use ofnucleases described above, and as is described in other sections of thisinvention, it is preferred to modify the oligonucleotides with theaddition of polypeptides or other molecules to improve and ease analysisand selectivity in the mass spectrometer. Because many restrictionendonucleases recognize palindromic sequences it is also possible toincrease the level of signal two-fold by the use of palindromicoligonucleotide substrates which form dimers. Each cleavage event formstwo identical products. Longer concatamers may also be produced creatinglarger, multi-mass-labeled substrate.

[0274] Antibodies are not the only possible target-recognition moleculethat may be used in these assays. Polypeptides derived from methods suchas phage display with target binding properties, as well as a variety ofnative proteins that demonstrate some binding activity of interest, maybe used instead. Targets may also be something other than proteins andcan include a variety of biologically relevant small molecules,including enzyme cofactors, hormones, neurotransmitters, and otherbiopolymers including polysaccharides and most importantly nucleicacids. Nucleic acid hybridization interactions may be used where boththe target and the recognition molecule are comprised of nucleic acids.Nucleic acids and other nonpeptide recognition molecules may be bound tothe enzyme involved in substrate conversion covalently via a variety oflinkage chemistries, some of which have been described here in the XXXsection, or noncovalently through a biotin/avidin linkage where theavidin is conjugated to the substrate conversion enzyme. One skilled inthe art can identify other linking methods.

[0275] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE 1 Synthesis of Peptide-Labeled Oligonucleotides

[0276] A. Preparation of Peptide-Linked Nucleoside 5′ Triphosphates

[0277] Preparation of peptide-linked nucleoside 5′-triphosphatesinvolves synthesis and coupling of allylamino-substituted dNTPs. Anexample is shown in FIG. 6A. 5-(3-aminoallyl)-2′-deoxyuridine5′-triphosphate (c) was prepared according to the procedure of Langer etal., 1981). Treatment of dUTP (a) with mercuric acetate at pH 5-7provides the 5-mercurated derivative (b). Allylation in the presence ofa palladium catalyst then provided c, which was coupled to the NHS-ester(d) of a suitably protected peptide (lysine and N-terminal aminesblocked with FMOC groups). Base deprotection of the peptide resulted information of the desired product (e). Alternatively, theallylamino-nucleotide (c) was treated sequentially with thehetero-bifunctional crosslinking reagent mal-sac-HNSA (Bachem BioscienceInc., King of Prussia, Pa.) and an N-terminal cysteine peptide to givethe conjugate (f).

[0278] B. Preparation of Peptide-Labeled Phosphoramidites

[0279] Peptide nucleoside phosphoramidite conjugates were prepared from5′-protected allylaminonucleosides as shown in FIG. 6B. Selectivedimethoxytritylation of uridine (h) provided the 5′-DMT ether (i), thatwas allylated via the mercurinucleoside with palladium catalyst (Dale etal., 1973; Langer et al., 1981). Treatment of the NHS-ester of asuitably protected peptide and conversion of the conjugate to thephosphoramidite (Sproat et al., 1987) provided the desired compound (k).

[0280] C. Synthesis of a 5′ Labeled Oligonucleotide-Peptide Conjugate

[0281] Oligonucleotide g (FIG. 6C) (SEQ ID NO:10) was prepared usingstandard solid-phase phosphoramidite chemistry. The5′-amino-modification through a disulfide linkage was achieved bysequential addition of Thio-Modifier C6 S-S and Amino-Modifier C6 dT(Glen Research Inc., Sterling, Va.) to the 5′-end. The oligonucleotidewas coupled to the heterobifunctional reagent mal-sac-HNSA (BachemCalifornia Inc., Torrance, Calif.) through the terminal primary aminogroup, purified by exclusion chromatography, and covalently coupled to apeptide with the sequence CGR GSG K through the N-terminal cysteinethiol. The conjugate was purified by ion-exchange chromatography, andanalyzed by MALDI-TOF mass spectrometry (FIG. 7X). The peck at m/z 8401in FIG. 7X corresponds to the desired conjugate.

[0282] D. Synthesis of a 3′ Labeled Oligonucleotide

[0283] A 3′ phosphorylated oligonucleotide with the sequence5′-TGAGGTGCGTGTTTGTGCCTGTp-3′ (SEQ ID NO: 1) was synthesized by standardphosphoramidite chemistry. A MALDI mass spectrum of the unconjugatedoligonucleotide is shown in FIG. 7A. The 3′-terminal T residue of theoligonucleotide was modified with a primary amino-group that wasincorporated during the synthesis as the modified phosphoramidite(C6-amino modifier, Glen Research Inc., Sterling, Va.). Theoligonucleotide was coupled through the active amino group to a peptideusing the hetero-bifunctional coupling reagent mal-sac-HNSA (BachemInc., Torrance, Calif.). The sequence of the peptide used for couplingto the oligonucleotide was CGYGPKKKRKVGG (SEQ ID NO: 2) (Sigma ChemicalCo., St. Louis, Mo.). The reaction to couple the peptide to theoligonucleotide occurs at the reactive thiol group on the N-terminalcysteine residue. After the coupling reaction, which is carried outaccording to standard procedure, the crude coupled product is purifiedby reversed phase HPLC. Fractions containing the desired coupled productwere identified by MALDI-MS, and were combined and evaporated todryness. The dried material was dissolved in a small amount of water andthe concentration determined by UV absorbance at 260 nm. A MALDI massspectrum of the oligonucleotide-peptide conjugate is shown in FIG. 7B.The major peak at m/z 8622.8 agrees well with desired product, while thepeck at 7051.7 is due to a residual amount of unreacted oligonucleotide(ca. 20%).

[0284] E. Synthesis of an Internally-Labeled Oligonucleotide-PeptideConjugate

[0285] An oligonucleotide of the sequence 5′-GGT TTA CAT GTT CCAA(aminoT)A TGA T-3′ (SEQ ID NO: 11) was prepared by standardphosphoramidite chemistry using Amino-Modifier C6 dT (Glen ResearchInc., Sterling, Va.) to incorporate the internal amino-modification. Theoligonucleotide was coupled to the hetrobifunctional reagentmal-sac-HNSA (Bachem California Inc., Torrance, Calif.) through theinternal primary amino group, purified by exclusion chromatography, andcovalently coupled to a peptide with the sequence CGT RGS GKG TG throughthe N-terminal cysteine thiol. The conjugate was purified byion-exchange chromatography, and analyzed by MALDI-TOF mass spectrometry(FIG. 7X). The peak at m/z 8075 in FIG. 7X corresponds to the desiredconjugate.

EXAMPLE 2 Detection of a Specific Target Sequence

[0286] As an example of the utility of the oligonucleotide-peptideconjugate as a probe in a hybridization study, a model system wasdesigned using a synthetic complementary strand as target DNA. A 42-merwas synthesized as a model target, with the sequence5′-CTCCCAGGACAGGCACAAACACGCACCTCAAAGCTGTTCCGT-3′ (SEQ ID NO: 3).Detection of the target was based on release of the peptide mass label(SEQ ID NO: 2) from the probe by a digestion with the 3′-5′double-strand-specific exonuclease III with analysis by MALDI-MS.

[0287] A mixture of 1 pmol of probe and 1 pmol of target in a 9 μLvolume of 1×Exonuclease III buffer (66 mM Tris-HCl, pH 8.0; 5 mM DTT;6.6 mM MgCl₂; 50 μg/mL BSA) was allowed to anneal by heating thesolution for 2 minutes in a boiling water bath and then slowly coolingit to room temperature over the course of about 20 minutes. ExonucleaseIII (USB, Cleveland, Ohio) was diluted from its stock concentration of17.5 U/μL in 1×buffer, and a 1 μL aliquot was added to the annealedtarget-probe solution. Four controls were included and runsimultaneously with the test solution. Control sample a contained bothtarget and probe but no exonuclease III, control sample B containedprobe and Exonuclease III but no target, control sample C containedprobe and Exonuclease III together with a random non-complementary36-mer, and control sample D contained only Exonuclease III. Themixtures were allowed to incubate for 30 minutes at room temperature. A1 μL aliquot of the solution was removed and added on top of apolycrystalline spot of 2.5-dihydroxybenzoic acid on a MALDI-MS sampleplate. The resulting positive-ion mass spectra of the test and controlsamples A, B and C are shown in FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D.Only the test sample in FIG. 8A showed a peak at 2045.3, the massexpected for the released peptide-nucleotide conjugate, demonstratingthat in this model system the inventors were able to specifically detectthe presence of the target sequence by a sensitive and rapid method.Selective Enzymatic cleavage of a Peptide. Oxidized bovine insulin chainB (Sigma Chemical Company, St. Louis, Mo.) in Tris•HCl (pH=7.8) wastreated with Endoproteinase Glu-C (w/w ratio 20:1, Sigma ChemicalCompany, St. Louis, Mo.) at 37° C. for 2 hours, and examined byMALDI-TOF mass spectrometry. The analysis (FIG. XX) indicated that theinsulin (SEQ ID NO: 12) was efficiently cleaved at the carboxyl side ofglutamyl residues into three fragments, m/z 1533 (FVNQHLC[SO₃H]GSHLVE)(SEQ ID NO: 13), m/z 1089 (RGFFYTPKA) (SEQ ID NO: 14), and m/z 919(ALYLVC[SO₃H]GE) (SEQ ID NO: 15). The relative intensities of the threepeaks in the mass spectrum reflect the number of basic (ionizable)functionalities in the three fragments. The largest molecular weightfragment contains two moderately basic histidine residues and istherefore only modestly visible in the spectrum. The middle fragmentcontains strongly basic lysine and arginine residues and thereforedisplays an intense peak. The smallest fragment has only the terminalamino-group available for protonation, and is therefore barelydetectable in the spectrum.

EXAMPLE 3 Detection of MRNA using Mass-Labeled Primers and rtPCR™

[0288] A pair of PCR™ priers for the ribosomal protein L7 gene wassynthesized by standard phosphoramidite chemistry with a modifiedamino-thymidine (Glen Research, Sterlin, Va.) incorporated near the3′-end of each. The sequence of the forward primer was5′-ATCTGAAGTCAGTAAAT*GAAC-3′ (SEQ ID NO: 4) and the sequence for thereverse primer was 5′-ATTTACCAGAGAT*CGAG-3′ (SEQ ID NO: 5), where T*represents the amino-modified thymidine. Each primer was mass-labeledwith a unique peptide by a standard coupling reaction between the aminogroup of the amino-modified thymidine and a sulfhydryl group on thepeptide through the heterobifunctional linker mal-SAC-HNSA (BachemCorp., Torrance, Calif.), and purified by ion-exchange HPLC. The peptidemass label used for the forward primer had the sequence CGYGPKKKRKVGG(SEQ ID NO: 2), and for the reverse primer the peptide wasCKNLNKDKQVYRATHR (SEQ ID NO: 6).

[0289] A reverse transcription reaction was performed on 10 μg of totalRNA isolated from a stable cancer cell line to generate first strandcDNA. The reaction was performed in a total volume of 20 μl andcontained 0.5 mg of oligo dT₁₅ primer (SEQ ID NO. 9) and 25 units of AMVreverse transcriptase. A PCR™ reaction was performed on 1 μ1 of thefirst strand cDNA using 10 pmol each of the forward and reversemass-labeled primers and 0.25 units of Taq DNA polymerase in a 10 μ1reaction. The rtPCR™ product was purified through a Microcon-30ultrafiltration unit (Amicon, Inc., Beverly, Mass.) according to themanufacturer's directions. After collecting the DNA from the filterunit, it was evaporated to dryness in a vacuum centrifuge andresuspended in 3.5 μ1 H₂O.

[0290] A digestion reaction using the double-strand specific 5′-3′exonuclease of T7 gene 6 was then performed. To the 3.5 μ1 of purifiedPCR™ product was added 0.5 μ1 of 10×buffer (660 mM Tris, pH 8, 6.6 mMMgCl₂) followed by 1 μ1 (5 units) of T7 gene 6 exonuclease (AmershamInc.). A control digestion was performed at the same time and contained5 units of enzyme, 5 pmol of free forward primer in an identical buffer.The digestion reactions were allowed to incubate at 37° C. for 60minutes followed by a heat inactivation of the enzyme (85° C. for 15minutes). A small portion of anion exchange resin (DEAE Sephadex A-25,Aldrich Chemical Co., Milwaukee, Wis.) was added to each digestion and a1 μ1 portion of the supernatant was removed and analyzed by MALDI-TOFmass spectrometry (positive ions, 3.5-dihydroxy benzoic acid matrix).The resulting mass spectra of the digested PCR™ product and control areshown in FIG. 15A and FIG. 15B respectively.

EXAMPLE 4 Detection of a Mixture of cDNA Plasmids

[0291] A mixture of 100 ng each of six and 50 ng of a seventhsingle-strand M13 plasmid clones, each containing unique inserts, wasdesalted and concentrated in a Microco-30 ultrafiltration unit accordingto the manufacturer's directions. The DNA, after collection, wasevaporated to dryness and resuspended in 1 μ1 of H₂O. A mixture of sevenmass-labeled probes containing 2.5 pmol each was added. Each probe wascomplementary to a portion of the insert for each clone in the mixtureand was coupled to a unique peptide mass label. The probes were allowedto hybridize by heating the mixture to 95° C. for 30 seconds followed bya 1 minute incubation at 45° C. After cooling the mixture to 37° C.,0.35 units of Exonuclease III was added and the digestion was allowed toproceed for 60 minutes. The reaction was allowed to cool to roomtemperature and then a small portion of DEAE Sephadex A25 anion exchangeresin (Aldrich Chemical Co., Milwaukee, Wis.) was added. A 1 μ1 portionof the supernatant was then removed and analyzed by MALDI-TOF massspectrometry (positive ions, 2.5-dihydroxy benzoic acid marix). Theresulting mass spectrum of the mixture of released mass labels is shownin FIG. 16.

EXAMPLE 5 SNP Analysis with Mass-labeled Primers and BiotinylatedDideoxyucleoside Triphosphates

[0292] A primer (“Primer A”) containing a chemically-releasable masslabel is synthesized and purified according to the method described inExample 1 C. Two synthetic template strands are also synthesized bystandard solid phase synthesis techniques. The sequence of Primer A is5′LTSS-GTGCTCAAGAACTACATGG-3′ (SEQ ID NO: 16) and the sequences for thetemplate strands are 5′TACTACCAGTTCCATGTAGTTCTTGAGCAC-3′ (Template 1T)(SEQ ID NO: 17) and 5′-TACTCCAGTACCATGTAGTTCTTGAGCAC-3′ (Template 1A)(SEQ ID NO: 18), where LT indicates the mass label attached to anamino-modified thymidine, SS represents the chemically cleavabledisulfide-containing group, and the boldface base designations in thetemplate strands indicate the polymorphic sites adjacent to the 3′-endof the primer. The primer is mass-labeled with a synthetic peptidepossessing the sequence CGRGSGK (SEQ ID NO: 19).

[0293] Two cycle-sequencing reactions are performed. Each reactioncontains 2 pmol of mass-labeled Primer A, 100 fmol of either Template 1Tor Template 1A, 200 pmol of Biotin-ddUTP (Boehringer-Mannheim, Inc.) and2.4 units of the thermostable DNA polymerase AmpliTaq-FS (Perkin-ElmerInc.) in a total volume of 20 μL. Both reactions are begun using typicalhot-start conditions. The reactions are performed according to thefollowing thermal cycling program: denaturing at 90° C. for 30 s,annealing at 50° C. for 10 s, extension at 65° C. for 10 s, for a totalof 35 cycles. Upon completion, the sequencing reactions are purified bycapturing the extended biotinylated products on streptavidin-coatedmagnetic beads. The beads are washed to remove unextended primer andthen the mass label released by treatment of the bead-bound product witha mild reducing agent to cleave the disulfide bond and release the masslabel into solution. A 1 μL portion of the supernatant is removed andanalyzed by MALDI-TOF mass spectrometry (positive ions, 2,5-dihydroxybenzoic acid matrix). The resulting mass spectra of the reactioncontaining the correct template to extend with biotin-ddUTP and of thereaction containing the incorrect template are shown in FIG. 17A andFIG. 17B, respectively. Since signal can only be seen in the spectrum inFIG. 17A as expected for the proper nucleotide incorporation, theseresults demonstrate the possibility of performing an SNP analysis usinga mass-labeled primer together with biotinylated dideoxynucleosidetriphosphates.

EXAMPLE 6 Multiplexed SNP Analysis with Mass-labeled Primers andBiotinylated Dideoxynucleoside Triphosphates

[0294] Two primers (“Primer B” and “Primer C”) each containing a uniquechemically-releasable mass label are synthesized and purified accordingto the method described in Example 1C. A synthetic template strand foreach is also synthesized by standard solid phase synthesis techniques.The sequence of the Primer B is 5′-LTSS-TCGGAGTCAACGGATTTG-3′ (SEQ IDNO: 20) and the sequence for the corresponding template strand is5′-TCCAGTTCTCAAATCCGTTGACTCCGA-3′ (“Template 2T”) (SEQ ID NO: 21).Primer C and its template strand (“Template 3T”) have the sequences5′-LTSS-GATGTCTGTATATGTTGCACTG-3′ (SEQ ID NO: 22) and5′-AAGTTGACTCTCAGTGCAACATATACAGACATC-3′ (SEQ ID NO: 23), respectively,where IT, SS, and boldface have the same meanings as described inExample 5. Primer B is mass-labeled with the synthetic peptideCAGGRGGGKGGA (SEQ ID NO: 24) and Primer C with the synthetic peptideCASGRGSGKGSA (SEQ ID NO: 25).

[0295] A multiplexed cycle-sequencing reaction is performed with PrimerA, Primer B, Primer C and each of the corresponding templates. Thereaction contains 2 pmol of each mass-labeled primer, 100 fmol each ofTemplate 1T, Template 2T and Template 3T, 200 pmol of Biotin-ddATP(Clonetech, Inc.) and 2.4 units of the thermostable DNA polymeraseAmpliTaq-FS (Perkin-Elmer Inc.) in a total volume of 20 μL. The reactionis begun using typical hot-start conditions and is performed accordingto the following thermal cycling program: denaturing at 90° C. for 30 s,annealing at 50° C. for 10 s, extension at 65° C. for 10 s, for a totalof 35 cycles. Upon completion, the sequencing reaction is purified bycapturing the extended biotinylated products on streptavidin-coatedmagnetic beads. The beads are washed to remove unextended primer andthen the mass labels released by treatment of the bead-bound productswith a mild reducing agent to cleave the disulfide bonds and release themass labels into solution. A 1 μL portion of the supernatant is removedand analyzed by MALDI-TOF mass spectrometry (positive ions,2,5-dihydroxy benzoic acid matrix). The resulting mass spectrum showingsignals for each of the expected mass-labels with peaks labeled as A, Band C referring to primers A, B and C respectively is shown in FIG. 18.This demonstrates the potential for performing multiplex SNP analysesutilizing mass-labeled primers.

EXAMPLE 7 SNP Analysis with Mass-labeled Primers and BiotinylatedNucleoside Triphosphates plus Normal Dideoxynucleoside Triphosphates

[0296] Two cycle-sequencing reactions are performed with primer a andone of either template 1T (SEQ ID NO: 17) or template 1A (SEQ ID NO:18). Each reaction contains 2 pmol of mass-labeled primer and 100 fmolof template. The triphosphates in each reaction consist of 200 pmol eachof Biotin-dCTP (Clonetech, Inc.), dATP and ddTTP. The reactions areperformed with 2.4 units of the thermostable DNA polymerase AmpliTaq-FS(Perkin-Elmer Inc.) in a total volume of 20 mL. The reactions are begunusing typical hot-start conditions and are performed according to thefollowing thermal cycling program: denaturing at 90° C. for 30 s,annealing at 50° C. for 10 s, extension at 65° C. for 10 s, for a totalof 35 cycles. Upon completion, the sequencing reactions are purified bycapturing the extended biotinylated products on streptavidin-coatedmagnetic beads. The beads are washed to remove unextended primer andthen the mass labels released by treatment of the bead-bound productswith a mild reducing agent to cleave the disulfide bonds and release themass labels into solution. A 1 mL portion of each supernatant is removedand analyzed by MALDI-TOF mass spectrometry (positive ions,2.5-dihydroxy benzoic acid matrix). The resulting mass spectra for thereaction containing template 1T and the reaction containing template 1Aare shown in FIG. 19A [XX3a] and FIG. 19B [XX3b], respectively.

EXAMPLE 8 Mass Label Tagging of Degenerate Base Primers and theIdentification of Sequence Variants by Extension with BiotinylatedDideoxynucleoside Triphosphates

[0297] Two primers related to Primer a and differing only in theidentity of the 3′-terminal base are synthesized and mass-labeledaccording to the method described in Example 1C. The sequence of PrimerD is 5′-LTSS-GTGCTCAAGAACTACATGA-3′ (SEQ ID NO: 26) and the sequence ofPrimer E is 5′-LTSS-GTGCTCAAGAACTACATGT-3′ (SEQ ID NO: 27), where LT andSS have the meanings described in Example 5. A synthetic template strand(“Template 4A”) is also synthesized using standard solid phase synthesistechniques. The sequence of the template strand is5′-TACTCCAGTTACATGTAGTTCTTGAGCAC-3′ (SEQ ID NO: 28), where the boldfaceindicates the base that varies from Template 1T. Primers D and E aremass-labeled with two unique synthetic peptide that differ from thepeptide attached to Primer A. The peptide attached to Primer D isCAGGRGGGKGGA (SEQ ID NO: 29), while the peptide attached to Primer E isCASGRGSGKGSA (SEQ ID NO: 30).

[0298] Two cycle-sequencing reactions are performed. Each reactioncontains 2 pmol each of mass-labeled Primer A, Primer D, and Primer E,100 fmol of either Template 1 T or Template 4A, 200 pmol of Biotin-ddATP(Clonetech, Inc.) and 2.4 units f the thermostable DNA polymeraseAmpliTaq-FS (Perkin-Elmer Inc.) in a total volume of 20 μL. Bothreactions are begun using typical hot-start conditions. The reactionsare performed according to the following thermal cycling program:denaturing at 90° C. for 30 s, annealing at 60° C. for 10 s, extensionat 65° C. for 10 s, for a total of 35 cycles. Upon completion, thesequencing reactions are purified by capturing the extended biotinylatedproducts on streptavidin-coated magnetic beads. The beads are washed toremove unextended primer and then the mass label released by treatmentof the bead-bound product with a mild reducing agent to cleave thedisulfide bound and release the mass label into solution. A 1 μL portionof the supernatant is removed and analyzed by MALDI-TOF massspectrometry (positive ions, 2.5-dihydroxy benzoic acid matrix). Theresulting mass spectra for the Primer E matched template and for thePrimer A matched template are shown in FIG. 20A and FIG. 20B,respectively. When Primer E is perfectly matched to the template, thepredominant mass label signal seen in the mass spectrum is that fromPrimer E. Likewise, when Primer a is perfectly matched to the templatein the reaction, the predominant mass label signal seen in the massspectrum is from Primer A. This example demonstrates the potential unityof using a mixture of degenerate, uniquely mass-labeled primers todetermine a variable sequence that is adjacent to a fixed sequence.

EXAMPLE 9 Single-Strand Selective Chemical Release of Mass Label

[0299] A chemically-cleavable oligonucleotide probe (SEQ ID NO: 31)containing a bridging 5′-S-P phosphodiester linkage in the backbone issynthesized by standard solid phase synthesis techniques incorporating amodified phosphoramidite reagent at the site of cleavage as described inPCT Patent Application WO 96/37630. The sequence of the 25-mer probe is5′-CCTGGCAAACTCAACTAGGC(sT)GTCC-3′ (SEQ ID NO: 31), where sT indicatesthe cleavage site. A complementary 35-mer oligonucleotide with thesequence 5′-GATCCGGACAGCCTAGTTGAGTTTGC-CAGGTAAGA-3′ (SEQ ID NO: 32), islikewise synthesized.

[0300] The probe and complement are hybridized together to form a duplexDNA in 1M triethylammonium acetate buffer by heating a mixture of 10pmol each at 95° C. for 3 min followed by a 10 min incubation at 70° C.and a subsequent 50° C. 10 min incubation. The mixture is allowed tocome to room temperature and AgNO₃ is added to a final concentration of0.14 mM. The silver promoted cleavage reaction is allowed to proceed for60 min at room temperature (20° C.) after which the reaction is quenchedby the addition of excess dithiothreitol. After evaporation of thesample, 3-HPA MALDI matrix solution is added to redissolve the DNA. Thesolution is spotted onto the mass spectrometer sample plate andanalyzed. The resulting mass spectrum and a mass spectrum of ano-complement control cleavage are shown in FIG. 21A and FIG. 21B,respectively. The spectrum of the control reaction shows that under theconditions used, the single-stranded oligonucleotide goes to about 90%complete cleavage, while the spectrum of the double-stranded form showsthat under identical conditions not more than about 5% cleavage occurs.This demonstrates the potential use a chemical cleavage reagent todiminiscrate between hybridized and unhybridized probes for release ofmass label.

EXAMPLE 10 Release of Mass Label by Exonuclease III Digestion of DNAProbe Hybridized to an RNA Transcript

[0301] A pair of PCR primers for the ribosomal protein L7 gene issynthesized by standard phosphoramidite chemistry. The forward primercontained at the 5′-end and extension which is the promoter region of T7RNA polymerase. The sequence of the forward primer is5′-TAATACGACTCACTATAGGGAGACTGCTGAGGATTGTA-GAGC-3′ (SEQ ID NO: 33) andthe sequence for the reverse primer is 5′-TCCAACAGTATAGATCTCATG-3′ (SEQID NO: 34). A pair of probes is also synthesized, each containing uniquemass labels. The probes are designed such that each hybridizes to adifferent strand of the PCR product while only one of them hybridizes toa strand of transcribed RNA. The peptide mass label used for theupper-strand probe had the sequence CGYGPKKKRKVGG (SEQ ID NO: 35), andfor the lower-strand (RNA-specific) probe the peptide wasCKNLNKDKQVYRATHRB (SEQ ID NO: 36). The synthesis of the mass-labeledprobes is described in Example 1E.

[0302] A reverse transcription reaction was performed on 10 μg of totalRNA isolated from a stable cancer cell line to generate first strandcDNA. The reaction was performed in a total volume of 20 μL andcontained 0.5 μg of oligo dTO primer and 25 units of AMV reversetranscriptase. A PCR reaction was performed on 1 μL of the first strandcDNA using 10 pmol each of the T7-forward and reverse primers and 1 unitof Taq DNA polymerase in a 20 μL reaction.

[0303] A two microliter aliquot of the RT-PCR product is then used for a20 microliter transcription reaction which contains 100 units of T7 RNApolymerase, 20 units of RNAsin inhibitor and 1 mM concentration of eachrNTP. The transcription reaction is allowed to proceed at 37° C. for 2h. One microliter of the transcription reaction product is then probedusing 5 pmol each of the two strand specific probes above. As a control,one microliter of the RT-PCR product is used instead of thetranscription reaction product. The probes and targets are hybridized in1×exonuclease III buffer by heating the mixture to 95° C. for 3 min,then incubating at 65° C. for 1 min then cooling to 37° C. ExonucleaseIII is then added to the mixture and the digestion is allowed to proceedat 37° C. for 1 h. A 1 μL portion of the supernatant was removed andanalyzed by MALDI-TOF mass spectrometry (positive ions, 2.5-dihydroxybenzoic acid matrix). The resulting mass spectra of the digested RNAtranscription product and control are shown in FIG. 22A and FIG. 22B,respectively. Only the RNA-strand specific probe mass label signal isseen in the transcription reaction sample while both probe mass labelsignals are seen when the RT-PCR product is probed. The fact that onlythe RNA-strand specific probe produces a signal in the mass spectrumwhen RNA transcript is present, together with the fact that signals fromboth probes should be seen if the signal were resulting only fromresidual RT-PCR product, shows that the enzyme exonuclease III can beused to specifically digest a probe hybridized to an RNA transcript torelease a mass label.

EXAMPLE 11 Matrix Selectivity for Peptide Mass Label or DNA

[0304] A 2 pmol portion of each of the mass-labeled primers Primer a andPrimer C is treated with a mild reducing agent to cleave the molecule atthe disulfide bond to yield separate peptide and DNA fragments. For eachprimer, a 1 microliter portion is spotted onto the mass spectrometersample plate with the matrix 2,5-dihydroxybenzoic acid, and a second 1microliter portion is spotted with the matrix 3-HPA. The mass spectrumfor Primer C obtained with 2.5-dihydroxybenzoic acid is shown in FIG.23A and shows a strong peptide signal with only very weak, poorlyresolved signal at the expected mass of the DNA fragment. In contrast,the mass spectrum obtained with 3-HPA (FIG. 23B) shows a strong, sharpsignal for the DNA fragment and a weaker signal for the peptidefragment. The corresponding spectra obtained for primer A are shown inFIG. 23C (2,5-DHB) and FIG. 23D (3-HPA). These results demonstrate thatit is possible to selectively detect a released mass-labeled section ofa probe in the presence of the much larger portion of the probe notcarrying a mass label.

EXAMPLE 12 Detection of a Specific Biomolecule (T) in a RestrictionEnzyme-linked Immunoadsorbent Assay

[0305] As an example of the detection of a target biomolecule viarelease of mass labels, a model system based on ELISA technology wasdesigned. This assay incorporates a DNA restriction enzyme for thedigestion of a mass-labeled substrate that is ultimately detected bymass spectrometry. This example describes a antibody-sandwich ELISA todetect soluble antigens. ELISA are described in Ausubel et al., (1997),incorporated herein by reference. Synthesis of the probe (mass labelbound to double-strand oligonucleotide containing an EcoRI restrictionsite) is described in Example 1. Double-stranded probe is prepared byhybridization of complementary oligonucleotides. Standard solutions ofantigen T are prepared for calibration of the assay (1-1000 ng/mL,depending on the linear range of the assay). Specific capture antibodies(Anti-T) and a target recognition molecule crosslinked to therestriction enzyme EcoRI (Anti T-EcoRI) are also prepared (0.1 units ofEcoRI per ng of specific antibody; 10 units per mL.).

Procedure

[0306] 1. Coat wells of microwell dishes (Immulon or equivalent) withthe capture antibody (10 ug/mL) which then is bound overnight accordingto the manufacturer's instructions. Block the residual binding capacityof the plate with blocking buffer (a buffered solution of 0.05% Tween 20and 0.25% bovine serum albumin) by filling wells with the solution andincubating 30 min at room temperature. Rinse plates with water threetimes and remove residual water.

[0307] 2. Bind solutions of known and unknown amounts of antigen T (inblocking buffer) to the wells, 50 μL/well and incubate at least 2 h.Wash plate three times with water, then treat with blocking buffer for10 min. Rinse again with water three times.

[0308] 3. Add 50 μL of anti T-EcoRI (containing 0.5 unit of EcoRIactivity) to each well and incubate 2 h at room temperature. Wash plate3 times using 1×ExoRI buffer containing 0.25% BSA.

[0309] 4. For each 96-well dish, mix

[0310] 140 μL Double-strand probe (10 pmol of mass-labeledoligonucleotide, 7 μM stock)

[0311] 100 μL EcoRI buffer (10×)

[0312] 760 μL H₂O

[0313] 5. Add 10 μL of the above mix to each well; incubate at 37° C.for the appropriate time to obtain a linear response with concentrationof T (up to 1 h). Heat inactivate enzyme at 65° C. for 20 min then coolto 4° C. Spot 1 μ1 of the mixture with DHB, wash dried spots 2× with 2μL of H₂O, and analyze for the released mass label.

[0314] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

References

[0315] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

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1 36 1 23 DNA Artificial Sequence Oligonucleotide 1 tgaggtgcgtgtttgtgcct gtn 23 2 13 PRT Artificial Sequence Oligopeptide 2 Cys GlyTyr Gly Pro Lys Lys Lys Arg Lys Val Gly Gly 1 5 10 3 42 DNA ArtificialSequence Oligonucleotide 3 ctcccaggac aggcacaaac acgcacctca aagctgttccgt 42 4 21 DNA Artificial Sequence Oligonucleotide 4 atctgaagtcagtaaangaa c 21 5 17 DNA Artificial Sequence Oligonucleotide 5atttaccaga gancgag 17 6 16 PRT Artificial Sequence Oligopeptide 6 CysLys Asn Leu Asn Lys Asp Lys Gln Val Tyr Arg Ala Thr His Arg 1 5 10 15 712 PRT Artificial Sequence Oligopeptide 7 Thr Cys Val Glu Trp Leu ArgArg Tyr Leu Lys Asn 1 5 10 8 16 PRT Artificial Sequence Oligopeptide 8Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ser Ala Asp 1 5 1015 9 15 DNA Artificial Sequence Oligonucleotide 9 tttttttttt ttttt 15 1022 DNA Artificial Sequence Oligonucleotide 10 ggtttacatg ttccaanatg at22 11 11 PRT Artificial Sequence Oligopeptide 11 Cys Gly Thr Arg Gly SerGly Lys Gly Thr Gly 1 5 10 12 30 PRT Artificial Sequence Oligopeptide 12Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 1015 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala 20 25 30 1313 PRT Artificial Sequence Oligopeptide 13 Phe Val Asn Gln His Leu CysGly Ser His Leu Val Glu 1 5 10 14 9 PRT Artificial Sequence Oligopeptide14 Arg Gly Phe Phe Tyr Thr Pro Lys Ala 1 5 15 8 PRT Artificial SequenceOligopeptide 15 Ala Leu Tyr Leu Val Cys Gly Glu 1 5 16 20 DNA ArtificialSequence Oligonucleotide 16 ngtgctcaag aactacatgg 20 17 29 DNAArtificial Sequence Oligonucleotide 17 tactccagtt ccatgtagtt cttgagcac29 18 29 DNA Artificial Sequence Oligonucleotide 18 tactccagtaccatgtagtt cttgagcac 29 19 7 PRT Artificial Sequence Oligopeptide 19 CysGly Arg Gly Ser Gly Lys 1 5 20 19 DNA Artificial SequenceOligonucleotide 20 ntcggagtca acggatttg 19 21 27 DNA Artificial SequenceOligonucleotide 21 tccagttctc aaatccgttg actccga 27 22 23 DNA ArtificialSequence Oligonucleotide 22 ngatgtctgt atatgttgca ctg 23 23 33 DNAArtificial Sequence Oligonucleotide 23 aagttgactc tcagtgcaac atatacagacatc 33 24 12 PRT Artificial Sequence Oligopeptide 24 Cys Ala Gly Gly ArgGly Gly Gly Lys Gly Gly Ala 1 5 10 25 12 PRT Artificial SequenceOligopeptide 25 Cys Ala Ser Gly Arg Gly Ser Gly Lys Gly Ser Ala 1 5 1026 20 DNA Artificial Sequence Oligonucleotide 26 ngtgctcaag aactacatga20 27 20 DNA Artificial Sequence Oligonucleotide 27 ngtgctcaagaactacatgt 20 28 29 DNA Artificial Sequence Oligonucleotide 28tactccagtt acatgtagtt cttgagcac 29 29 12 PRT Artificial SequenceOligopeptide 29 Cys Ala Gly Gly Arg Gly Gly Gly Lys Gly Gly Ala 1 5 1030 12 PRT Artificial Sequence Oligopeptide 30 Cys Ala Ser Gly Arg GlySer Gly Lys Gly Ser Ala 1 5 10 31 25 DNA Artificial SequenceOligonulceotide 31 cctggcaaac tcaactaggc ngtcc 25 32 35 DNA ArtificialSequence Oligonulceotide 32 gatccggaca gcctagttga gtttgccagg taaga 35 3342 DNA Artificial Sequence Oligonulceotide 33 taatacgact cactatagggagactgctga ggattgtaga gc 42 34 21 DNA Artificial SequenceOligonulceotide 34 tccaacagta tagatctcat g 21 35 13 PRT ArtificialSequence Oligopeptide 35 Cys Gly Tyr Gly Pro Lys Lys Lys Arg Lys Val GlyGly 1 5 10 36 16 PRT Artificial Sequence Oligopeptide 36 Cys Lys Asn LeuAsn Lys Asp Lys Gln Val Tyr Arg Ala Thr His Arg 1 5 10 15

What is claimed is:
 1. A release tag compound comprising Rx, Re and M wherein: Rx is a reactive group; Re is a release group; and M is a nonvolatile mass label comprising a synthetic polymer or a biopolymer detectable by mass spectrometry.
 2. The compound of claim 1, wherein the mass label comprises a biopolymer.
 3. The compound of claim 2, wherein the biopolymer comprises one or more monomer units, wherein each monomer unit is separately and independently selected from the group consisting of an amino acid, a nucleic acid, and a saccharide.
 4. The compound of claim 3, wherein each monomer comprises an amino acid.
 5. The compound of claim 3, wherein each monomer comprises a nucleic acid.
 6. The compound of claim 1, wherein the mass label comprises a synthetic polymer.
 7. The compound of claim 6, wherein the synthetic polymer comprises polyethylene glycol, polyvinyl phenol, polypropylene glycol, polymethyl methacrylate, and derivatives thereof.
 8. The compound of claim 7, wherein the synthetic polymer comprises polyethylene glycol.
 9. The compound of claim 1, wherein the release group comprises a chemically cleavable linkage.
 10. The compound of claim 9, wherein the chemically cleavable linkage comprises a modified base, a modified sugar, a disulfide bond, a chemically cleavable group incorporated into the phosphate backbone, or a chemically cleavable linker.
 11. The compound of claim 10, wherein the chemically cleavable linkage further comprises a moiety cleavable by acid, base, oxidation, reduction, heat, light, metal ion catalyzed, displacement, or elimination chemistry.
 12. The compound of claim 10, wherein the chemically cleavable group is incorporated into the phosphate backbone.
 13. The compound of claim 12, wherein the chemically cleavable group comprises dialkoxysilane, 3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate, 3′-(N)-phosphoroamidate, or 5′-(N)-phosphoroamidate.
 14. The compound of claim 10, wherein the chemically cleavable linkage comprises a modified sugar.
 15. The compound of claim 14, wherein the modified sugar comprises ribose.
 16. The compound of claim 10, wherein the chemically cleavable linkage comprises a disulfide bond.
 17. The compound of claim 1, wherein the reactive group comprises a biomolecule capable of specific molecular recognition.
 18. The compound of claim 17, wherein the biomolecule capable of specific molecular recognition comprises a polypeptide.
 19. The compound of claim 18, wherein the polypeptide is selected from the group consisting essentially of an antibody, an enzyme, a receptor, a regulatory protein, a nucleic acid-binding protein, a hormone, and a protein product of a display method.
 20. The compound of 19, wherein the polypeptide comprises a product of a phage display method or a bacterial display method.
 21. The compound of claim 10, wherein the polypeptide comprises an antibody or an enzyme.
 22. The compound of claim 17, wherein the biomolecule capable of specific molecular recognition comprises a polynucleic acid.
 23. The compound of claim 22, wherein the polynucleic acid comprises an oligonucleotide.
 24. The compound of claim 1, wherein Rx and Re are the same.
 25. The compound of claim 1, wherein Re is contained within Rx.
 26. The compound of claim 1, wherein Re is cleavable by an enzyme.
 27. The compound of claim 26, wherein Re is a phosphodiester or amide linkage.
 28. The compound of claim 26, wherein the enzyme comprises a nuclease
 29. The compound of claim 28, wherein the nuclease comprises an exonuclease.
 30. The compound of claim 29, wherein the exonuclease is specific for double-stranded polynucleic acids.
 31. The compound of claim 29, wherein the exonuclease is specific for single-stranded polynucleic acids.
 32. The compound of claim 28, wherein the nuclease comprises a restriction endonuclease.
 33. The compound of claim 32, wherein the restriction endonuclease comprises a Type IIS restriction endonuclease.
 34. The compound of claim 32, wherein the restriction endonuclease comprises a Type II restriction endonuclease.
 35. The compound of claim 27, wherein the enzyme comprises a protease.
 36. The compound of claim 35, wherein the protease comprises an endoproteinase.
 37. The compound of claim 1, wherein more than one mass label is incorporated.
 38. The compound of claim 1, wherein the mass label has a molecular weight greater than about 500 Daltons.
 39. A release tag compound comprising Rx, Re and M, wherein: Rx is a reactive group comprising mass-labeled nucleotides; Re is a release group; and M is a mass label detectable by mass spectrometry.
 40. A release tag compound comprising Rx, Re and M, wherein: Rx is a reactive group comprising nucleoside phosphoramidites; Re is a release group; and M is a mass label detectable by mass spectrometry.
 41. A release tag compound comprising Rx, Re and M, wherein: Rx is a reactive group comprising a first oligonucleotide having a nucleotide or a second oligonucleotide attached thereto; Re is a release group; and M is a mass label detectable by mass spectrometry.
 42. The compound of claim 41, wherein the nucleotide added after hybridization comprises a chain terminating modification.
 43. The compound of claim 41, wherein the nucleotide or second oligonucleotide further comprise a functional group capable of being immobilized on a solid support.
 44. The compound of claim 43, wherein the functional group comprises a biotin, or digoxigenin.
 45. A release tag compound comprising Rx, Re and M, wherein: Rx is an oligonucleotide comprising a nuclease blocking moiety; Re is a release group; and M is a mass label detectable by mass spectrometry.
 46. The compound of claim 45, wherein the nuclease blocking moiety is selected from the group consisting essentially of a phosphorothioate, an alkylsilydiester, a boranophosphate, a methylphosphonate, and a peptide nucleic acid.
 47. A release tag compound comprising Rx, Re and M, wherein: Rx is a double-stranded oligonucleotide comprising a restriction endonuclease recognition site; Re is a release group comprising a phosphodiester linkage capable of being cleaved by a restriction endonuclease; and M is a mass label detectable by mass spectrometry.
 48. The compound of claim 47, wherein Rx further comprises a modified nucleotide.
 49. The compound of claim 48, wherein comprises a portion of Rx.
 50. The compound of claim 47, wherein Rx further comprises a self-complementary oligonucleotide hairpin.
 51. A release tag compound comprising Rx, Re and M, wherein: Rx is a double-stranded oligonucleotide; Re is a chemically cleavable release group; and M is a mass label detectable by mass spectrometry; wherein Re is located within Rx; and wherein cleavage at the chemically cleavable release group is inhibited by the presence of a double-stranded oligonucleotide at said release group.
 52. The compound of claim 51, wherein the chemically cleavable release group comprises 3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate, 3′-(N)-phosphoroamidate, or 5′-(N)-phosphoroamidate, or ribose.
 53. The compound of claim 51, wherein hybridization of a portion of Rx to a target nucleic acid renders a portion of Rx single-stranded at Re.
 54. A set of release tag compounds, said set comprising one or more release tag compounds for detecting a particular target nucleic acids, each release tag compound comprising Rx, Re and M, wherein: Rx is an oligonucleotide comprising a variable region and an invariant region; Re is a release group; and M is a mass label detectable by mass spectrometry; wherein said invariant and variable regions react with the target nucleic acid.
 55. The set of claim 54, wherein the mass label of at least one release tag compound identifies a specific sequence within the variable region.
 56. The set of claim 55, wherein the mass label for each release tag compound uniquely identifies a different sequence within the variable region.
 57. The set of claim 54, wherein a combination of the mass labels of two or more release tag compounds identifies a different sequence within the variable region.
 58. The set of claim 54, wherein Rx further comprises a nucleotide or oligonucleotide attached thereto after hybridization to the target nucleic acid.
 59. The set of claim 58, wherein the added nucleotide or oligonucleotide comprises Re′ and M′, wherein: Re′ is a release group; and M′ is a mass label detectable by mass spectrometry.
 60. The set of claim 58, wherein the added nucleotide comprises a chain terminating moiety.
 61. The set of claim 58, wherein the added nucleotide or oligonucleotide further comprises a functional group capable of being immobilized on a solid support.
 62. The set of claim 61, wherein the functional group comprises a biotin or digoxigenin.
 63. A method for multiplexing the detection of a target molecule comprising: (a) obtaining a plurality of probes, each comprising a compound of claim 1; (b) contacting the target molecule with the plurality of probes to produce probe:target molecule complexes, wherein the target molecule is attached to the reactive group of the probe; (c) releasing the mass labels from the probe:target molecule complexes to produce released mass labels; and (d) determining the mass of the released mass labels by mass spectrometry, wherein each reactive group in a probe:target molecule complex is associated with a unique set of mass labels.
 64. The method of claim 63, wherein a plurality of target molecules is contacted with the plurality of probes.
 65. The method of claim 63, wherein the set of mass labels are attached to the same probe.
 66. The method of claim 63, wherein the set of mass labels are attached to different probes.
 67. The method of claim 63, wherein the target molecule is immobilized onto a solid support.
 68. The method of claim 66, wherein a plurality of target molecules are immobilized onto the solid support at spaced locations.
 69. The method of claim 63, wherein the target molecule is selected from the group consisting essentially of a polynucleotide, an antigen, a ligand, a polypeptide, a carbohydrate, and a lipid.
 70. The method of claim 69, wherein the target molecule comprises a polynucleotide.
 71. The method of claim 69, wherein the target molecule comprises a polypeptide.
 72. The method of claim 63, wherein the reactive group comprises a biomolecule capable of specific molecular recognition.
 73. The method of claim 72, wherein the reactive group comprises a polypeptide.
 74. The method of claim 72, wherein the polypeptide is selected from the group consisting essentially of an antibody, an enzyme, a receptor, a regulatory protein, a nucleic-acid-binding protein, a hormone, or a protein product of a display methods.
 75. The method of claim 74, wherein the polypeptide comprises an antibody.
 76. The method of claim 74, wherein the polypeptide comprises a product of a phage display method or a bacterial display method.
 77. The method of claim 72, wherein the reactive group comprises a polynucleotide or an oligonucleotide.
 78. The method of claim 63, wherein the unique set of mass labels comprises a mass label that indicates the presence of a specified component within the reactive group.
 79. The method of claim 78, wherein the mass label indicates the presence of the specified component at a specified location within the reactive group.
 80. The method of claim 79, wherein a reactive group comprising n specified components is associated with a unique set of mass labels having (n) members.
 81. The method of claim 80, wherein (n) is from 1 to
 1000. 82. The method of claim 79, wherein a reactive group comprising n specified components is associated with a unique set of mass labels having y members wherein n is less than y!/; and wherein x comprises the number of mass labels used per active group.
 83. The method of claim 79, wherein the plurality of probes each comprise a known reactive group having a known set of mass labels.
 84. The method of claim 83, wherein the plurality of probes are prepared by combinatorial synthesis.
 85. The method of claim 79, wherein the plurality of target molecules comprise a known chemical structure.
 86. The method of claim 63, wherein the target molecule comprises an expressed gene product.
 87. The method of claim 86, wherein the expressed gene product is derived from a cloned mRNA.
 88. The method of claim 63, wherein the target molecule comprises a cloned genomic DNA.
 89. A method of monitoring gene expression comprising: (a) obtaining a plurality of probes, each probe comprising a compound of claim 1; (b) obtaining a plurality of target nucleic acids with the plurality of probes to produce probe:target nucleic acid complexes; (c) selectively releasing the mass labels from the probe:target nucleic acid complexes, wherein the complexes are in solution; and (d) determining the mass of the released mass labels by mass spectrometry.
 90. The method of claim 89, herein the target nucleic acids comprise mRNA or first-strand cDNA.
 91. The method of claim 89, wherein the target nucleic acids comprise amplified nucleic acid products.
 92. The method of claim 89, wherein the amplification is effected by PCR, rtPCR, LCR, Qbeta Replicase, SDA, CPR, TAS, NASBA, or multiple rounds of RNA transcription or some combination thereof.
 93. A method of monitoring gene expression comprising: (a) obtaining an mRNA pool; (b) amplifying a subset of the mRNA pool to produce a plurality of amplified nucleic acid products; (c) obtaining a plurality of probes, each probe comprising a compound of claim 1; (d) contacting the plurality of amplified nucleic acid products with the plurality of probes to produce probe; amplified nucleic acid product complexes; (e) selectively releasing the mass label from the probe:amplified nucleic acid product complexes to produce released mass labels; and (f) determining the mass of the released mass labels by mass spectrometry.
 94. The method of claim 93, wherein at least one probe is capable of being immobilized onto a solid support.
 95. The method of claim 93, wherein at least one amplified nucleic acid product is capable of being immobilized onto a solid support.
 96. A method for detecting a target molecule, said method comprising the steps of: (a) contacting a target molecule with a probe comprising a compound of claim 1 to produce probe:target molecule complexes and unreacted probes; (b) releasing the mass labels from the probe:target molecule complexes to produce released mass labels; (c) selectively desorbing the released mass label from an organic matrix to produce desorbed mass labels; and (d) determining the mass of the desorbed mass label by mass spectrometry.
 97. The method of claim 96, wherein the organic matrix comprises 2,5-dihydroxybenzoic acid, sinapinic acid, or alpha-cyano-4-hydroxycinnamic acid.
 98. A method for detecting the presence of a target nucleic acid molecule, said method comprising: (a) obtaining a probe comprising a compound of claim 1; (b) contacting the probe to a target nucleic acid molecule to produce probe:nucleic acid molecule complexes; (c) mass modifying the probe:nucleic acid molecule complexes by attaching a nucleotide or oligonucleotide to the probe to produce mass modified mass labels; (d) releasing the mass modified mass labels; and (e) determining the mass of the mass-modified mass labels by mass spectrometry.
 99. A method for detecting specific biomolecules in an enzyme-linked affinity assay comprising (a) obtaining a substrate; (b) contacting the target molecule with an affinity ligand-enzyme conjugate to produce an affinity ligand-enzyme conjugate:target molecule complex; (c) contacting the affinity ligand-enzyme conjugate:target molecule complex with the substrate to produce a mass modified product; and (d) determining the mass of the mass modified product by mass spectrometry.
 100. The method of claim 99, wherein the enzyme is a restriction endonuclease.
 101. The method of claim 100, further comprising a plurality of restriction endonuclease conjugates.
 102. The method of claim 100, wherein the substrate comprises a restriction site.
 103. The method of claim 99, wherein the affinity ligand is a biomolecule capable of specific molecular recognition.
 104. The method of claim 103, wherein the affinity ligand is a polypeptide.
 105. The method of claim 104, wherein the polypeptide is selected from the group consisting essentially of an antibody, an enzyme, a receptor, a regulatory protein, a nucleic acid-binding protein, a hormone, or a protein product of a display method.
 106. The method of claim 105, wherein the polypeptide comprises a product of a phage display method or a bacterial display method.
 107. The method of claim 105, wherein the polypeptide comprises an antibody.
 108. The method of claim 103, wherein the affinity ligand is a polynucleic acid.
 109. The method of claim 99, wherein the enzyme is a protease.
 110. The method of claim 109, wherein the substrate is a polypeptide.
 111. A method of determining the identity of a nucleotide at a single nucleotide polymorphism site, the method comprising the steps of: (a) obtaining at least one probe comprising a compound of claim 1 with a reactive group capable of hybridizing to a target nucleic acid containing a single nucleotide polymorphism site; (b) hybridizing the probe to the target nucleic acid to produce a probe:target nucleic acid complex; (c) selectively releasing a mass label to obtain a released mass label, and; (d) determining the mass of the released mass label by mass spectrometry, wherein the identity of the nucleotide at the single nucleotide polymorphism site corresponds to the mass of the released mass label.
 112. The method of claim 111, wherein the probe is obtained by hybridizing the reactive group to the target nucleic acid to produce a reactive group:target nucleic acid complex and extending the reactive group by the addition of one or more added nucleotides.
 113. The method of claim 112, wherein at least one added nucleotide is mass-labeled.
 114. The method of claim 111, further comprising a set of probes having an invariant region and a variant region, wherein the invariant region is capable of hybridizing to a region immediately adjacent to a single nucleotide polymorphism site and wherein at least one member of the set of probes contains a variable region complementary to the nucleotide at the single nucleotide polymorphism site.
 115. A method of determining the identity of a plurality of nucleotides at a plurality of single nucleotide polymorphism sites, the method comprising the steps of: (a) obtaining a plurality of probes each comprising a reactive group capable of hybridizing to a target nucleic acid containing a single nucleotide polymorphism site; (b) hybridizing the probes to one or more target nucleic acids to produce one or more probe:target nucleic acid complexes; (c) selectively releasing a plurality of mass labels to obtain released mass labels, and; (d) determining the mass of the released mass labels by mass spectrometry, wherein the identity of the nucleotide at each single nucleotide polymorphism site corresponds to the mass of a released mass label.
 116. The method of claim 115, wherein one or more probes are obtained by hybridizing a reactive group to a target nucleic acid to produce a reactive group:target nucleic acid complex and extending the reactive group by the addition of one or more added nucleotides.
 117. The method of claim 116, wherein at least one added nucleotide is mass-labeled.
 118. The method of claim 115, further comprising a set of probes having an invariant region and a variant region, wherein the invariant region is capable of hybridizing to a region immediately adjacent to a single nucleotide polymorphism site and wherein at least one member of the set of probes contains a variable region complementary to the nucleotide at the single nucleotide polymorphism site.
 119. The method of detecting a single-stranded nucleic acid target molecule, said method comprising the steps of: (a) obtaining a double-stranded nucleic acid probe comprising a reactive group, a release group and a nonvolatile mass label, wherein the double-stranded probe further comprises a complementary strand and a mass-labeled strand, and wherein the mass-labeled strand further comprises a nonvolatile mass label and a release group; (b) obtaining a single-stranded target molecule; (c) contacting the single-stranded target with the double-stranded probe to produce a complementary strand:target molecule complex and a single stranded mass-labeled strand; (d) selectively releasing the mass label from the single-stranded mass-labeled strand; (e) determining the mass of the mass label by mass spectrometry.
 120. A method for multiplexing the detection of at least one single stranded target molecule, said method comprising the steps of: (a) obtaining a plurality of double-stranded probes, each comprising a compound of claim 1, wherein each of the double-stranded probes further comprise a complementary strand and a mass-labeled strand, and wherein the mass-labeled strand further comprises a nonvolatile mass label and a release group; (b) obtaining at least one single-stranded target molecule; (c) contacting the target molecule with the plurality of probes to produce a complementary strand:target molecule complex and a single stranded mass-labeled strand; (d) selectively releasing the mass label from the single-stranded mass-labeled strand; (e) determining the mass of the mass label by mass spectrometry, wherein each reactive group of the double-stranded probe is associated with a unique set of mass labels.
 121. A method of preparing a release tag compound, the method comprising: (a) obtaining a target nucleic acid sequence; (b) synthesizing a reactive group capable of hybridizing to the target nucleic acid sequence; (c) hybridizing the reactive group to the target nucleic acid sequence; and (d) extending the reactive group to produce a release tag compound complementary to the target nucleic acid sequence, wherein the release tag compound comprises a reactive group and release group and a mass-labeled detectable by mass spectrometry.
 122. The method of claim 121, further comprising amplifying the release tag compound by polymerase chain reaction. 